13093077
Second harmonic generation microscopy reveals altered collagen microstructure in usual interstitial pneumonia versus healthy lung
BackgroundIt is not understood why some pulmonary fibroses such as cryptogenic organizing pneumonia (COP) respond well to treatment, while others like usual interstitial pneumonia (UIP) do not. Increased understanding of the structure and function of the matrix in this area is critical to improving our understanding of the biology of these diseases and developing novel therapies. The objectives herein are to provide new insights into the underlying collagen- and matrix-related biological mechanisms driving COP versus UIP.MethodsTwo-photon second harmonic generation (SHG) and excitation fluorescence microscopies were used to interrogate and quantify differences between intrinsic fibrillar collagen and elastin matrix signals in healthy, COP, and UIP lung.ResultsCollagen microstructure was different in UIP versus healthy lung, but not in COP versus healthy, as indicated by the ratio of forward-to-backward propagating SHG signal (FSHG/BSHG). This collagen microstructure as assessed by FSHG/BSHG was also different in areas with preserved alveolar architecture adjacent to UIP fibroblastic foci or honeycomb areas versus healthy lung. Fibrosis was evidenced by increased col1 and col3 content in COP and UIP versus healthy, with highest col1:col3 ratio in UIP. Evidence of elastin breakdown (i.e. reduced mature elastin fiber content), and increased collagen:mature elastin ratios, were seen in COP and UIP versus healthy.ConclusionsFibrillar collagen’s subresolution structure (i.e. “microstructure”) is altered in UIP versus COP and healthy lung, which may provide novel insights into the biological reasons why unlike COP, UIP is resistant to therapies, and demonstrates the ability of SHG microscopy to potentially distinguish treatable versus intractable pulmonary fibroses.
15222858
Cryptogenic organising pneumonia
Organising pneumonia is defined histopathologically by intra-alveolar buds of granulation tissue, consisting of intermixed myofibroblasts and connective tissue. Although nonspecific, this histopathological pattern, together with characteristic clinical and imaging features, defines cryptogenic organising pneumonia when no cause or peculiar underlying context is found. Rapid clinical and imaging improvement is obtained with corticosteroid treatment, but relapses are common after stopping treatment.
43896808
Interstitial and Vascular Type V Collagen Morphologic Disorganization in Usual Interstitial Pneumonia
Recent evidence suggests that type V collagen plays a role in organizing collagen fibrils, thus maintaining fibril size and spatial organization uniform. In this study we sought to characterize the importance of type V collagen morphological disorganization and to study the relationship between type V collagen, active remodeling of the pulmonary vascular/parenchyma (fibroblastic foci), and other collagen types in usual interstitial pneumonia (UIP). We examined type V collagen and several other collagens in 24 open lung biopsies with histological pattern of UIP from patients with idiopathic pulmonary fibrosis (IPF). We used immunofluorescence, morphometry, and three-dimensional reconstruction to evaluate the amount of collagen V and its interaction with the active remodeling progression in UIP, as well as types I and III collagen fibers. Active remodeling progression was significantly related to type V collagen density (p<0.05), showing a gradual and direct increase to minimal, moderate, and severe fibrosis degree in UIP and in the three different areas: normal, intervening, and mural-organizing fibrosis in UIP. Parenchymal changes were characterized by morphological disorganization of fibrillar collagen with diverse disarray and thickness when observed by three-dimensional reconstruction. We concluded that in the different temporal stages of UIP, vascular/parenchyma collagen type V is increased, in disarray, and is the most important predictor of survival.
7958245
Abnormal deposition of collagen/elastic vascular fibres and prognostic significance in idiopathic interstitial pneumonias
Background: Vascular remodelling has recently been shown to be a promising pathogenetic indicator in idiopathic interstitial pneumonias (IIPs). Aim: To validate the importance of the collagen/elastic system in vascular remodelling and to study the relationships between the collagen/elastic system, survival and the major histological patterns of IIPs. Methods: Collagen/elastic system fibres were studied in 25 patients with acute interstitial pneumonia/diffuse alveolar damage, 22 with non-specific interstitial pneumonia/non-specific interstitial pneumonia and 55 with idiopathic pulmonary fibrosis/usual interstitial pneumonia. The Picrosirius polarisation method and Weigert’s resorcin–fuchsin histochemistry and morphometric analysis were used to evaluate the amount of vascular collagen/elastic system fibres and their association with the histological pattern of IIPs. The association between vascular remodelling and the degree of parenchymal fibrosis in usual interstitial pneumonia (UIP) was also considered. Results: The vascular measurement of collagen/elastic fibres was significantly higher in UIP than in the lungs of controls, and in those with diffuse alveolar damage and those with non-specific interstitial pneumonia. In addition, the increment of collagen/elastic fibres in UIP varied according to the degree and activity of the parenchymal fibrosis. The most important predictors of survival in UIP were vascular remodelling classification and vascular collagen deposition. Conclusion: A progressive vascular fibroelastosis occurs in IIP histological patterns, probably indicating evolutionarily adapted responses to parenchymal injury. The vascular remodelling classification and the increase in vascular collagen were related to survival in IIP and possibly play a role in its pathogenesis. Further studies are needed to determine whether this relationship is causal or consequential.
25426035
Impact of angiotensin-converting enzyme inhibitors and statins on survival in idiopathic pulmonary fibrosis.
STUDY OBJECTIVES
To assess the clinical relevance of angiotensin-converting enzyme inhibitors (ACEI) and 3-hydroxy-3-methylglutaryl coenzyme-A reductase inhibitors (statins) in the context of idiopathic pulmonary fibrosis (IPF).


BACKGROUND
IPF is a progressive interstitial lung disease for which there is no effective treatment. ACEI and statins have been shown to possess antifibrotic properties in experimental models in vitro and in vivo.


DESIGN, SETTING, AND PATIENTS
Retrospective review of the effects of ACEI and statins on survival of 478 patients with IPF seen at Mayo Clinic Rochester from 1994 through 1996. Fifty-two patients (11%) were receiving ACEI, 35 patients (7%) were receiving statins, and 5 patients (1%) patients were receiving both at their initial visit.


RESULTS
For subjects receiving ACEI, the median survival from the index visit was 2.2 years, compared to 2.9 years for subjects not receiving ACEI (p = 0.088). The median survival was 2.9 years if patients were receiving statins or not (p = 0.573). There was also no significant difference in survival between patients with IPF receiving either ACEI or statins vs those receiving neither at the index visit (2.5 years vs 3 years, respectively; p = 0.066). After adjusting for age, gender, recommended IPF treatment, smoking status, prior oxygen use, FVC, diffusion capacity for carbon monoxide, coronary artery disease, congestive heart failure, diabetes mellitus, and hypertension, there were no differences in survival between those subjects receiving either ACEI, statins, or both vs neither.


CONCLUSIONS
These data do not suggest a beneficial effect of ACEI and/or statins on survival in patients with IPF.
24510832
Natural history of idiopathic pulmonary fibrosis.
Idiopathic pulmonary fibrosis (IPF) is a parenchymal lung disease characterized by progressive interstitial fibrosis. IPF has a poor prognosis, with a median survival time of 2-3 years from diagnosis, but varying from a few months to a decade. The natural history of IPF is highly variable and the course of disease in an individual patient is difficult to predict. Some patients with IPF experience rapid decline, others progress much more slowly, and some patients show periods of relative stability interspersed with acute deteriorations in respiratory function. Many clinical, radiographic, serologic, and histopathologic variables have been shown to predict mortality in IPF. However, the accuracy of these predictors varies due to the retrospective nature of some of the studies and variations in study design. The ability to identify clinical characteristics that predict disease progression and survival would be useful for counseling patients, treatment decision-making, and prompt consideration for lung transplantation. A number of indices for predicting mortality in patients with IPF are available, but they require further validation. As high-resolution computed tomography scans become more widely available and patients with IPF are diagnosed earlier, survival times following diagnosis will improve. Early referral to interstitial lung disease specialty centers is important for accurate diagnosis and may be associated with improved outcomes. The goal of this review is to examine the natural history of IPF, discuss predictors of mortality, and highlight the importance of prompt diagnosis and referral for patients with IPF.
33394821
Recent Evidence for Pharmacological Treatment of Idiopathic Pulmonary Fibrosis
Objective: To describe emerging evidence for the pharmacological treatment of idiopathic pulmonary fibrosis (IPF). Data Sources: A search of PubMed (1966 to July 2014) was performed using the terms idiopathic pulmonary fibrosis and treatment. Study Selection and Data Extraction: Review of articles was restricted to articles in English and relating to placebo-controlled or comparative clinical trial data of recent significance. Evidence statements from the most recent international guidelines and some historical trial data were also included for context. Data Synthesis: Numerous treatment options have been evaluated for IPF. Therapies evaluated in large trials have either resulted in increased mortality (anticoagulation, triple-therapy with N-acetylcysteine [NAC], azathioprine, and prednisone) or demonstrated a lack of efficacy (endothelin receptor antagonists, single-agent NAC). Pirfenidone, a novel antifibrotic and anti-inflammatory agent, has demonstrated efficacy in several recent analyses and is the only approved medication for the treatment of IPF in more than 30 countries outside of the United States, with resubmission to the Food and Drug Administration (FDA) recently made. Nintedanib, a tyrosine kinase inhibitor, has demonstrated encouraging results in phase III studies and has also recently been submitted for FDA approval. Conclusions: Limited options have existed for the treatment of IPF. New evidence suggests that safe and efficacious treatment options for IPF are on the horizon in the form of pirfenidone and nintedanib, although both agents await FDA decisions.
41871394
Long-term clinical and real-world experience with pirfenidone in the treatment of idiopathic pulmonary fibrosis
Idiopathic pulmonary fibrosis (IPF) is a chronic, irreversible, progressively destructive lung disease that culminates in respiratory failure and death. Randomised controlled trials have demonstrated that treatment of IPF patients with pirfenidone reduces lung function decline, improves progression-free survival and significantly reduces the risk of all-cause mortality at 1 year. Pirfenidone has been shown to have a favourable safety profile and was generally well tolerated over the long term in clinical trials and real-world experience. However, side-effect management is critical to help some patients remain on treatment over the long term. The primary treatment-related adverse events associated with pirfenidone therapy are gastrointestinal upset, rash and photosensitivity. Gastrointestinal events may be mitigated by ensuring that pirfenidone is taken with food, while skin symptoms may be reduced by avoiding sun exposure and frequent use of sunblock. Educating patients about the potential for these adverse events to occur and providing instructions prior to treatment to avoid adverse drug reactions are an important means of ensuring patients may derive the important benefits provided by long-term treatment with pirfenidone. Patient education is critical to help IPF patients manage side-effects of pirfenidone and remain on treatment http://ow.ly/HaPak
28736508
Idiopathic pulmonary fibrosis: An update
Abstract Idiopathic pulmonary fibrosis (IPF) is the most common and lethal form of idiopathic interstitial pneumonia. The disease, which occurs primarily in middle-aged and older adults, is thought to arise following an aberrant reparative response to alveolar epithelial cell injury characterized by secretion of excessive amounts of extracellular matrix components, resulting in scarring of the lung, architectural distortion, and irreversible loss of function. A complex interplay between environmental and host factors is thought to contribute to the development of the disease, although the cause of IPF remains elusive and its pathogenesis incompletely understood. Over the last decade, disease definition and diagnostic criteria have evolved significantly, and this has facilitated the design of a number of high-quality clinical trials evaluating novel therapeutic agents for IPF. This massive effort of the medical and industry community has led to the identification of two compounds (pirfenidone and nintedanib) able to reduce functional decline and disease progression. These promising results notwithstanding, IPF remains a major cause of morbidity and mortality and a largely unmet medical need. A real cure for this devastating disease has yet to emerge and will likely consist of a combination of drugs targeting the plethora of pathways potentially involved in disease pathogenesis.
207488505
Pirfenidone: A Review of Its Use in Idiopathic Pulmonary Fibrosis
Pirfenidone (Esbriet®) is an orally administered, synthetic, pyridone compound that is approved for the treatment of adults with mild to moderate idiopathic pulmonary fibrosis (IPF) in the EU, and for the treatment of IPF in the USA. This article summarizes pharmacological, efficacy and tolerability data relevant to the use of pirfenidone in these indications. In the randomized, double-blind, placebo-controlled, multinational CAPACITY trials in patients with mild to moderate IPF, a significant reduction in the rate of decline in forced vital capacity (FVC) was seen with pirfenidone versus placebo in study 004 but not in study 006. Pirfenidone also reduced the rate of decline in FVC to a significantly greater extent than placebo in the randomized, double-blind, multinational ASCEND trial in this patient population. In addition, pirfenidone showed a significant treatment effect on the 6-min walking test distance and progression-free survival in the ASCEND trial and in a pooled analysis of the CAPACITY trials. Pirfenidone had a manageable tolerability profile in all three studies. Gastrointestinal and skin-related events (e.g. nausea, rash, photosensitivity reaction), which were the most commonly occurring treatment-emergent adverse events, were generally mild to moderate in severity. In addition, a prespecified mortality analysis across all three studies demonstrated a significant reduction in IPF-related and all-cause mortality with pirfenidone. In conclusion, oral pirfenidone is a valuable agent for use in patients with IPF.
32192389
Pharmacologic therapies for idiopathic pulmonary fibrosis, past and future
Abstract Idiopathic pulmonary fibrosis (IPF) is a severe, progressive fibrotic disease of the lung of unknown etiology that affects approximately 150,000 patients in the United States. It carries a median survival of two to three years, but clinical course can vary markedly from patient to patient. There has been no established treatment for IPF, but recent advances in coordinated clinical trials through groups such as IPFnet and academia–industry partnerships have allowed this relatively rare disease to be studied in much greater depth. Historically, the default therapy for IPF was a combination of prednisone, N-acetylcysteine, and azathioprine, but recent trials have shown that this regimen actually increases mortality. An enormous body of work in recent years, spanning the bench to the bedside, has radically altered our understanding of the molecular mechanisms underlying IPF. Newer modalities, particularly those involving monoclonal antibodies targeted at specific pathways known to contribute to the fibrotic process, have generated a great deal of excitement in the field, and recent clinical trials on therapies such as pirfenidone and nintedanib herald a new era in targeted IPF therapies.
40423214
Cryptogenic organizing pneumonia.
Cryptogenic organizing pneumonia is a rare, distinct disorder that is sufficiently different from the other diseases in the group of idiopathic interstitial pneumonias to be designated as a separate entity. In its most typical presentation, it is characterized by dyspnea and cough, with multiple patchy alveolar opacities on pulmonary imaging. Definite diagnosis is obtained by the finding of buds of granulation tissue in the distal airspaces at lung biopsy. No cause (as infection, drug reaction, or associated disease as connective tissue disease) is found. Corticosteroid treatment is rapidly effective, but relapses are common on reducing or stopping treatment.
205718456
Recent advances and new opportunities in lung mechanobiology.
Lung function is inextricably linked to mechanics. On short timescales every breath generates dynamic cycles of cell and matrix stretch, along with convection of fluids in the airways and vasculature. Perturbations such airway smooth muscle shortening or surfactant dysfunction rapidly alter respiratory mechanics, with profound influence on lung function. On longer timescales, lung development, maturation, and remodeling all strongly depend on cues from the mechanical environment. Thus mechanics has long played a central role in our developing understanding of lung biology and respiratory physiology. This concise review focuses on progress over the past 5 years in elucidating the molecular origins of lung mechanical behavior, and the cellular signaling events triggered by mechanical perturbations that contribute to lung development, homeostasis, and injury. Special emphasis is placed on the tools and approaches opening new avenues for investigation of lung behavior at integrative cellular and molecular scales. We conclude with a brief summary of selected opportunities and challenges that lie ahead for the lung mechanobiology research community.
5749258
Biomechanical regulation of mesenchymal cell function
Purpose of reviewCells of mesenchymal origin are strongly influenced by their biomechanical environment. They also help to shape tissue architecture and reciprocally influence tissue mechanical environments through their capacity to deposit, remodel, and resorb extracellular matrix and to promote tissue vascularization. Although mechanical regulation of cell function and tissue remodeling has long been appreciated in other contexts, the purpose of this review is to highlight the increasing appreciation of its importance in fibrosis and hypertrophic scarring. Recent findingsExperiments in both animal and cellular model systems have demonstrated pivotal roles for the biomechanical environment in regulating myofibroblast differentiation and contraction, endothelial barrier function and angiogenesis, and mesenchymal stem cell fate decisions. Through these studies, a better understanding of the molecular mechanisms transducing the biomechanical environment is emerging, with prominent and interacting roles recently identified for key network components including transforming growth factor-&bgr;/SMAD, focal adhesion kinase, MRTFs, Wnt/&bgr;-catenin and YAP/TAZ signaling pathways. SummaryProgress in understanding biomechanical regulation of mesenchymal cell function is leading to novel approaches for improving clinical outcomes in fibrotic diseases and wound healing. These approaches include interventions aimed at modifying the tissue biomechanical environment, and efforts to target mesenchymal cell activation by, and reciprocal interactions with, the mechanical environment.
41540512
Matrices of physiologic stiffness potently inactivate idiopathic pulmonary fibrosis fibroblasts.
Fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) have been shown to differ from normal lung fibroblasts in functional behaviors that contribute to the pathogenesis of IPF, including the expression of contractile proteins and proliferation, but how such behaviors vary in matrices with stiffness matched to normal and fibrotic lung tissue remains unknown. Here, we tested whether pathologic changes in matrix stiffness control IPF and normal lung tissue-derived fibroblast functions, and compared the relative efficacy of mechanical cues to an antifibrotic lipid mediator, prostaglandin E(2) (PGE(2)). Fibroblasts were grown on collagen I-coated glass or hydrogel substrates of discrete stiffnesses, spanning the range of normal and fibrotic lung tissue. Traction microscopy was used to quantify contractile function. The CyQuant Cell Proliferation Assay (Invitrogen, Carlsbad, CA) was used to assess changes in cell number, and PGE(2) concentrations were measured by ELISA. We confirmed differences in proliferation and PGE(2) synthesis between IPF and normal tissue-derived fibroblasts on rigid substrates. However, IPF fibroblasts remained highly responsive to changes in matrix stiffness, and both proliferative and contractile differences between IPF and normal fibroblasts were ablated on physiologically soft matrices. We also confirmed the relative resistance of IPF fibroblasts to PGE(2), while demonstrating that decreases in matrix stiffness and the inhibition of Rho kinase both potently attenuate contractile function in IPF-derived fibroblasts. We conclude that pathologic changes in the mechanical environment control important IPF fibroblast functions. Understanding how mechanical cues control fibroblast function may offer new opportunities for targeting these cells, even when they are resistant to antifibrotic pharmacological agents or biological mediators.
28902071
Lung parenchymal mechanics.
The lung parenchyma comprises a large number of thin-walled alveoli, forming an enormous surface area, which serves to maintain proper gas exchange. The alveoli are held open by the transpulmonary pressure, or prestress, which is balanced by tissues forces and alveolar surface film forces. Gas exchange efficiency is thus inextricably linked to three fundamental features of the lung: parenchymal architecture, prestress, and the mechanical properties of the parenchyma. The prestress is a key determinant of lung deformability that influences many phenomena including local ventilation, regional blood flow, tissue stiffness, smooth muscle contractility, and alveolar stability. The main pathway for stress transmission is through the extracellular matrix. Thus, the mechanical properties of the matrix play a key role both in lung function and biology. These mechanical properties in turn are determined by the constituents of the tissue, including elastin, collagen, and proteoglycans. In addition, the macroscopic mechanical properties are also influenced by the surface tension and, to some extent, the contractile state of the adherent cells. This chapter focuses on the biomechanical properties of the main constituents of the parenchyma in the presence of prestress and how these properties define normal function or change in disease. An integrated view of lung mechanics is presented and the utility of parenchymal mechanics at the bedside as well as its possible future role in lung physiology and medicine are discussed.
2964500
Collagen structure and stability.
Collagen is the most abundant protein in animals. This fibrous, structural protein comprises a right-handed bundle of three parallel, left-handed polyproline II-type helices. Much progress has been made in elucidating the structure of collagen triple helices and the physicochemical basis for their stability. New evidence demonstrates that stereoelectronic effects and preorganization play a key role in that stability. The fibrillar structure of type I collagen-the prototypical collagen fibril-has been revealed in detail. Artificial collagen fibrils that display some properties of natural collagen fibrils are now accessible using chemical synthesis and self-assembly. A rapidly emerging understanding of the mechanical and structural properties of native collagen fibrils will guide further development of artificial collagenous materials for biomedicine and nanotechnology.
9707181
Two-Photon and Second Harmonic Microscopy in Clinical and Translational Cancer Research
Application of two-photon microscopy (TPM) to translational and clinical cancer research has burgeoned over the last several years, as several avenues of pre-clinical research have come to fruition. In this review, we focus on two forms of TPM—two-photon excitation fluorescence microscopy, and second harmonic generation microscopy—as they have been used for investigating cancer pathology in ex vivo and in vivo human tissue. We begin with discussion of two-photon theory and instrumentation particularly as applicable to cancer research, followed by an overview of some of the relevant cancer research literature in areas that include two-photon imaging of human tissue biopsies, human skin in vivo, and the rapidly developing technology of two-photon microendoscopy. We believe these and other evolving two-photon methodologies will continue to help translate cancer research from the bench to the bedside, and ultimately bring minimally invasive methods for cancer diagnosis and treatment to therapeutic reality.
13550090
Stromal matrix metalloprotease-13 knockout alters Collagen I structure at the tumor-host interface and increases lung metastasis of C57BL/6 syngeneic E0771 mammary tumor cells
BackgroundMatrix metalloproteases and collagen are key participants in breast cancer, but their precise roles in cancer etiology and progression remain unclear. MMP13 helps regulate collagen structure and has been ascribed largely harmful roles in cancer, but some studies demonstrate that MMP13 may also protect against tumor pathology. Other studies indicate that collagen’s organizational patterns at the breast tumor-host interface influence metastatic potential. Therefore we investigated how MMP13 modulates collagen I, a principal collagen subtype in breast tissue, and affects tumor pathology and metastasis in a mouse model of breast cancer.MethodsTumors were implanted into murine mammary tissues, and their growth analyzed in Wildtype and MMP13 KO mice. Following extraction, tumors were analyzed for collagen I levels and collagen I macro- and micro-structural properties at the tumor-host boundary using immunocytochemistry and two-photon and second harmonic generation microscopy. Lungs were analyzed for metastases counts, to correlate collagen I changes with a clinically significant functional parameter. Statistical analyses were performed by t-test, analysis of variance, or Wilcoxon-Mann–Whitney tests as appropriate.ResultsWe found that genetic ablation of host stromal MMP13 led to: 1. Increased mammary tumor collagen I content, 2. Marked changes in collagen I spatial organization, and 3. Altered collagen I microstructure at the tumor-host boundary, as well as 4. Increased metastasis from the primary mammary tumor to lungs.ConclusionsThese results implicate host MMP13 as a key regulator of collagen I structure and metastasis in mammary tumors, thus making it an attractive potential therapeutic target by which we might alter metastatic potential, one of the chief determinants of clinical outcome in breast cancer. In addition to identifying stromal MMP13 is an important regulator of the tumor microenvironment and metastasis, these results also suggest that stromal MMP13 may protect against breast cancer pathology under some conditions, a finding with important implications for development of chemotherapies directed against matrix metalloproteases.
122398716
Second harmonic properties of tumor collagen: determining the structural relationship between reactive stroma and healthy stroma.
We utilize the polarization and directionality of light emitted by fibrillar collagen via second harmonic generation to determine structural relationships between collagen in mouse mammary tumor models and the healthy mammary fat pad. In spite of the aberrations in collagen production and degradation that are the hallmarks of tumor stroma, we find that the characteristic angle of SHG scatterers within collagen fibrils, and the spatial extent over which they are appropriately ordered for SHG production, are the same in tumor and healthy collagen. This suggests that the SHG-producing subpopulation of collagen is unaffected by the altered collagen synthesis of the tumor stroma, and protected from its aberrant degradative environment.
28657368
Phase Matching considerations in Second Harmonic Generation from tissues: Effects on emission directionality, conversion efficiency and observed morphology.
We present a heuristic treatment which relates SHG image intensities, signal directionality, and observed morphology to the physical structure of collagen and cellulose fibrillar tissues. The SHG creation model is based upon relaxed phase matching conditions which account for dispersion, randomness, and axial momentum contributions from the media, and includes a mathematical treatment which relates SHG conversion efficiency to fibril diameter and packing through the inclusion of potential intensity amplification resultant from quasi-phase matching (QPM). A direct consequence of this theory is that SHG in biological tissues is not strictly a coherent process, and that the forward directed SHG has a longer coherence length than the backward component, Through this treatment, we show that the emission directionality and also conversion efficiency do not arise solely from the fibril size but also depend on packing density and order of the inter-fibril structure. We demonstrate these principles in comparing the SHG response in normal and Osteogenesis Imperfecta (OI) skin. We show that the observed directionality and decreased relative intensity in the diseased state is consistent with phase matching conditions arising from the decreased fibril size and more random assembly. We further use this theory to explain the differences in morphology seen in forward and backward collected SHG in fibrillar tissues (e.g., collagenous and cellulosic). Specifically, we attribute segmented appearance to destructive interference between small fibrils separated by less than the coherence length. We suggest the approach based on relaxed phasematching conditions is general in predicting the SHG response in tissues and may be broadly applicable in interpreting the SHG contrast for diagnostic applications.
120363244
Second-harmonic generation by focused excitation of inhomogeneously distributed scatterers
We generalize a simple model describing second-harmonic generation (2HG) by highly focused excitation light to allow the possibility of sample inhomogeneities. Depending on their spatial frequencies these inhomogeneities can significantly modify the 2HG radiation patterns and total powers, and in certain cases provoke backward 2HG propagation. Examples are considered where the distribution of 2HG radiators is axially periodic or spherically localized, illustrating the effects of sample structure or clustering. Our model is specifically applicable to 2HG microscopy geometries.
22408819
Structure of type I and type III heterotypic collagen fibrils: an X-ray diffraction study.
The molecular packing arrangement within collagen fibrils has a significant effect on the tensile properties of tissues. To date, most studies have focused on homotypic fibrils composed of type I collagen. This study investigates the packing of type I/III collagen molecules in heterotypic fibrils of colonic submucosa using a combination of X-ray diffraction data, molecular model building, and simulated X-ray diffraction fibre diagrams. A model comprising a 70-nm-diameter D- (approximately 65 nm) axial periodic structure containing type I and type III collagen chains was constructed from amino acid scattering factors organised in a liquid-like lateral packing arrangement simulated using a classical Lennard-Jones potential. The models that gave the most accurate correspondence with diffraction data revealed that the structure of the fibril involves liquid-like lateral packing combined with a constant helical inclination angle for molecules throughout the fibril. Combinations of type I:type III scattering factors in a ratio of 4:1 gave a reasonable correspondence with the meridional diffraction series. The attenuation of the meridional intensities may be explained by a blurring of the electron density profile of the D period caused by nonspecific or random interactions between collagen types I and III in the heterotypic fibril.
23806401
Type I and Type III Collagen Interactions during Fibrillogenesis a
There is some evidence that type I and type III collagens may be present in the same fibril. In order to demonstrate this, double labeling immunofluorescence microscopy and immunoelectron microscopy were performed with antibodies directed against the collagen molecule and the aminopropeptide domains of type I and type III procollagens using embryonic (postabortion) and adult human skin. Double indirect and protein A immunoelectron microscopy were carried out with 5- and 15-nm gold particles. Skin extracts were also studied by immunoblotting. Double immunofluorescence microscopy with antibodies against type I and type III collagen molecules revealed patterns of fluorescence that were identical in both fetal and adult skins. Immunofluorescence microscopy using an antibody directed against the aminopropeptide of type III procollagen labeled the entire dermis in both embryonic and adult skins. In contrast, although the aminopropeptide of type I procollagen was present throughout embryonic dermis, it was markedly reduced in adult dermis, except for the epidermo-dermal junction. Double immunoelectron microscopy of fetal skin revealed labeling of the aminopropeptide of type I and type III procollagens on the same thin (20-30 nm) fibrils. Large type I fibrils (90-100 nm) were coated with type III collagen molecules and their corresponding aminopropeptide but not with the aminopropeptide of type I procollagen. The aminopropeptide of type I procollagen was present on thin fibrils only at the epidermo-dermal junction in adult skin. Immunoblotting of skin extracts revealed the presence of both pN-type III procollagen (collagen plus the aminopropeptide) and pN-type I procollagen in fetal skin, but only pN-type III in adult skin. This study demonstrates that type I and type III collagens coexist within the same fibril and that the aminopropeptide of type III procollagen is present at the surface of type I collagen fibrils that apparently have reached full growth.
13235841
Copolymerization of pNcollagen III and collagen I. pNcollagen III decreases the rate of incorporation of collagen I into fibrils, the amount of collagen I incorporated, and the diameter of the fibrils formed.
Previous observations suggested that pNcollagen III, the partially processed form of type III procollagen, coats fibrils of collagen I and thereby helps regulate the diameter of fibrils formed by collagen I. The previous observations, however, did not exclude the possibility that pNcollagen III was deposited on preformed collagen I fibrils after the fibrils were assembled. Here, mixtures of pNcollagen III and collagen I were generated simultaneously by enzymatic cleavage of precursor forms of the proteins. The results demonstrated that pNcollagen III forms true copolymers with collagen I. The presence of pNcollagen III both inhibited the rate at which collagen I assembled into fibrils and decreased the amount of collagen I incorporated into fibrils at steady-state equilibrium. In addition, the results demonstrated that copolymerization of pNcollagen III with collagen I generated fibrils that were thinner than fibrils generated under the same conditions from collagen I alone. Increasing the initial molar ratio of pNcollagen III to collagen I in the solution-phase increased the amount of pNcollagen III copolymerizing with collagen I and progressively decreased the diameter of the fibrils. Therefore, the copolymers were heterogeneous in that the stoichiometry of the two monomers in the fibrils varied. The results are consistent with a model in which pNcollagen III can regulate the diameter of collagen I fibrils by coating the surface of the fibrils and thereby allow tip growth but not lateral growth of the fibrils.
5204530
Second harmonic generation reveals matrix alterations during breast tumor progression
Abstract. Alteration of the extracellular matrix in tumor stroma influences efficiency of cell locomotion away from the primary tumor into surrounding tissues and vasculature, thereby affecting metastatic potential. We study matrix changes in breast cancer through the use of second harmonic generation (SHG) of collagen in order to improve the current understanding of breast tumor stromal development. Specifically, we utilize a quantitative analysis of the ratio of forward to backward propagating SHG signal (F/B ratio) to monitor collagen throughout ductal and lobular carcinoma development. After detection of a significant decrease in the F/B ratio of invasive but not in situ ductal carcinoma compared with healthy tissue, the collagen F/B ratio is investigated to determine the evolution of fibrillar collagen changes throughout tumor progression. Results are compared with the progression of lobular carcinoma, whose F/B signature also underwent significant evolution during progression, albeit in a different manner, which offers insight into varying methods of tissue penetration and collagen manipulation between the carcinomas. This research provides insights into trends of stromal reorganization throughout breast tumor development.
45601412
Second-harmonic generation scattering directionality predicts tumor cell motility in collagen gels
Abstract. Second-harmonic generation (SHG) allows for the analysis of tumor collagen structural changes throughout metastatic progression. SHG directionality, measured through the ratio of the forward-propagating to backward-propagating signal (F/B ratio), is affected by collagen fibril diameter, spacing, and disorder of fibril packing within a fiber. As tumors progress, these parameters evolve, producing concurrent changes in F/B. It has been recently shown that the F/B of highly metastatic invasive ductal carcinoma (IDC) breast tumors is significantly different from less metastatic tumors. This suggests a possible relationship between the microstructure of collagen, as measured by the F/B, and the ability of tumor cells to locomote through that collagen. Utilizing in vitro collagen gels of different F/B ratios, we explored the relationship between collagen microstructure and motility of tumor cells in a “clean” environment, free of the myriad cells, and signals found in in vivo. We found a significant relationship between F/B and the total distance traveled by the tumor cell, as well as both the average and maximum velocities of the cells. Consequently, one possible mechanism underlying the observed relationship between tumor F/B and metastatic output in IDC patient samples is a direct influence of collagen structure on tumor cell motility.
22972157
Quantification of collagen I in airway tissues using second harmonic generation
Abstract. Extracellular matrix (ECM) remodeling contributes to the pathogenic changes in chronic obstructive pulmonary disease (COPD) and is both complex and not well understood. Collagen I, a component of the ECM altered in COPD airways, has second harmonic generation (SHG) properties. The SHG signal is coherent, propagating both forward (F) (primarily organized/mature collagen fibrils) and backward (B) (primarily disorganized/immature collagen fibrils) parallel to the incident light. The F/B SHG ratio was used to determine the proportion of organized to disorganized collagen, with lower variation in F/B ratio between sampling regions within the same patient and between patients in the same disease group compared with analyzing F and B data alone. The F/B ratio was independent of laser power drift, regions analyzed within a tissue and tissue orientation during analysis. Using this method, we identified a significant difference in collagen organization in airway tissue between COPD and nondiseased. We have developed a robust optimization and calibration methodology that will allow direct comparison of data obtained at different times and from multiple microscopes, which is directly adaptable for use with other tissue types. We report a powerful new tool for advancing our understanding of pathological ECM remodeling that may uncover new therapeutic targets in the future.
19108010
Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation
The content and structure of collagen is essential in governing the delivery of therapeutic molecules in tumors. Thus, simple histological staining of tumor tissue biopsies for collagen could be used to assess the accessibility of molecular therapeutics in tumors. Here we show that it is possible to optically image fibrillar collagen in tumors growing in mice using second-harmonic generation (SHG). Using this noninvasive technique, we estimated relative diffusive hindrance, quantified the dynamics of collagen modification after pharmacologic intervention and provided mechanistic insight into improved diffusive transport induced by the hormone relaxin. This technology could offer basic scientists and clinicians an enhanced ability to estimate the relative penetrabilities of molecular therapeutics.
1074147
In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy
Edward B. Brown, Robert B. Campbell, Yoshikazu Tsuzuki, Lei Xu, Peter Carmeliet, Dai Fukumura & Rakesh K. Jain Nature Med. 7, 864–868 (2001). Some symbols and units in the following paragraph on page 868 were incorrect . The correct version is below. LE interactions and RBC velocity measurements. LE interactions were monitored in 100 × 300-μm windows with reduced spatial resolution (∼0.
33751891
Collagen and myosin characterization by orientation field second harmonic microscopy.
Collagen and myosin fibrils are endogenous harmonophores that both give rise to Second Harmonic Generation (SHG). By combining four polarization SHG images provided by a scanning microscope, we show that the orientation of the principal axis of the nonlinear susceptibility tensor chi(2) can be determined for each pixel of the image. The ratio rho = chi33/chi15 of the principal components of chi(2) of collagen and myosin was obtained with the same method, and found within the range 1.6-1.8 and 0.5-0.6 respectively. The orientation of the principal axis of chi(2) is shown to be correlated to the orientation of the fibrils themselves. This provides a straightforward method, which we call Orientation Field-Second Harmonic Microscopy (OF-SHM), to reconstruct orientation fields of fibrils at various scales and resolutions in different biological systems (from muscle sarcomere to the whole embryo).
4376227
Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres.
Several biologically important protein structures give rise to strong second-harmonic generation (SHG) in their native context. In addition to high-contrast optical sections of cells and tissues, SHG imaging can provide detailed structural information based on the physical constraints of the optical effect. In this study we characterize, by biochemical and optical analysis, the critical structures underlying SHG from the complex muscle sarcomere. SHG emission arises from domains of the sarcomere containing thick filaments, even within nascent sarcomeres of differentiating myocytes. SHG from isolated myofibrils is abolished by extraction of myosin, but is unaffected by removal or addition of actin filaments. Furthermore, the polarization dependence of sarcomeric SHG is not affected by either the proportion of myosin head domains or the orientation of myosin heads. By fitting SHG polarization anisotropy readings to theoretical response curves, we find an orientation for the elemental harmonophore that corresponds well to the pitch of the myosin rod alpha-helix along the thick filament axis. Taken together, these data indicate that myosin rod domains are the key structures giving SHG from striated muscle. This study should guide the interpretation of SHG contrast in images of cardiac and skeletal muscle tissue for a variety of biomedical applications.
35574640
Determination of collagen nanostructure from second-order susceptibility tensor analysis.
A model is proposed to describe the polarization dependence of second harmonic generation (SHG) from type I collagen fibrils. The model is based on sum-frequency vibrational spectrum experiments that attribute the molecular origins of collagen second-order susceptibility to the peptide groups in the backbone of the collagen α-helix and the methylene groups in the pyrrolidine rings. Applying our model to a polarization SHG (P-SHG) experiment leads to a predicted collagen I peptide pitch-angle of 45.82° ± 0.46° and methylene pitch-angle of 94.80° ± 0.97°. Compared to a previous model that accounts for only the peptide contribution, our results are more consistent with the x-ray diffraction determination of collagen-like peptide. Application of our model to type II collagen from rat trachea cartilage leads to similar results. The peptide pitch-angle of 45.72° ± 1.17° is similar to that of type I collagen, but a different methylene pitch-angle of 97.87° ± 1.79° was found. Our work demonstrates that far-field P-SHG measurements can be used to extract molecular structural information of collagen fibers.
792064
Estimation of helical angles of myosin and collagen by second harmonic generation imaging microscopy.
We performed Second Harmonic Generation (SHG) imaging microscopy of endogeneous myosin-rich and collagen-rich tissues in amphibian and mammals. We determined the relative components of the macroscopic susceptibility tensor chi((2)) from polarization dependence of SHG intensity. The effective orientation angle theta(e) of the harmonophores has been determined for each protein. For myosin we found theta(e) approximately 62 degrees and this value was unchanged during myofibrillogenesis. It was also independent of the animal species (xenopus, dog and human). For collagen we found theta(e) approximately 49 degrees for both type I- and type III- rich tissues. From these results we localized the source of SHG along the single helix of both myosin and collagen.
18141866
Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation
Multicolor nonlinear microscopy of living tissue using two- and three-photon-excited intrinsic fluorescence combined with second harmonic generation by supermolecular structures produces images with the resolution and detail of standard histology without the use of exogenous stains. Imaging of intrinsic indicators within tissue, such as nicotinamide adenine dinucleotide, retinol, indoleamines, and collagen provides crucial information for physiology and pathology. The efficient application of multiphoton microscopy to intrinsic imaging requires knowledge of the nonlinear optical properties of specific cell and tissue components. Here we compile and demonstrate applications involving a range of intrinsic molecules and molecular assemblies that enable direct visualization of tissue morphology, cell metabolism, and disease states such as Alzheimer's disease and cancer.
2013639
Minimally invasive multiphoton and harmonic generation imaging of extracellular matrix structures in lung airway and related diseases.
Multiphoton microscopy has become a powerful imaging method for minimally invasive evaluation of extracellular matrix (ECM) and cellular structures deep within tissues in their native environments. This technology, which uses ultra-short femto-second laser pulses as the excitation source, is efficient in multiphoton excitation fluorescence (MPEF) of endogenously fluorescent macromolecular systems and induction of highly specific second harmonic generation (SHG) signals from non-centrosymmetric macromolecules such as fibrillar collagens. Both these signals can be captured simultaneously to provide spatially resolved 3D structural organization of ECM as well as cellular morphologies in lung or airway tissue with spectral specificity and sensitivity. These imaging modalities are minimally invasive since structures deep within tissues can be visualized without the need for tissue fixation and/or sectioning. Much of the traditional histological and chemical procedures associated with conventional microscopy methods, which may alter native structure of lung tissue samples, can be circumvented to generate more accurate 3D morphological and fine structural information. In addition to outlining basic principles associated with MPEF and SHG microscopy methods, this review reports potential uses of these high resolution imaging modalities in lung structural imaging. We place special emphasis on imaging 3D structural features of airways, visualizing and quantifying ECM remodeling associated with mouse asthma model as well as the potential uses for multiphoton microscopy in in vitro airway applications.
6430042
Lung Parenchymal Mechanics in Health and Disease
The mechanical properties of lung tissue are important determinants of lung physiological functions. The connective tissue is composed mainly of cells and extracellular matrix, where collagen and elastic fibers are the main determinants of lung tissue mechanical properties. These fibers have essentially different elastic properties, form a continuous network along the lungs, and are responsible for passive expiration. In the last decade, many studies analyzed the relationship between tissue composition, microstructure, and macrophysiology, showing that the lung physiological behavior reflects both the mechanical properties of tissue individual components and its complex structural organization. Different lung pathologies such as acute respiratory distress syndrome, fibrosis, inflammation, and emphysema can affect the extracellular matrix. This review focuses on the mechanical properties of lung tissue and how the stress-bearing elements of lung parenchyma can influence its behavior.
72053
Toward a kinetic theory of connective tissue micromechanics.
The aim of this study is to develop unifying concepts at the microstructural level to account for macroscopic connective tissue dynamics. We establish the hypothesis that rate-dependent and rate-independent dissipative stresses arise in the interaction among fibers in the connective tissue matrix. A quantitative theoretical analysis is specified in terms of geometry and material properties of connective tissue fibers and surrounding constituents. The analysis leads to the notion of slip and diffusion boundary layers, which become unifying concepts in understanding mechanisms that underlie connective tissue elasticity and energy dissipation during various types of loading. The complex three-dimensional fiber network is simplified to the interaction of two ideally elastic fibers that dissipate energy on slipping interface surfaces. The effects of such interactions are assumed to be expressed in the aggregate matrix. Special solutions of the field equations are obtained analytically, whereas the general solution of the model field equations is obtained numerically. The solutions lead to predictions of tissue behavior that are qualitatively, if not quantitatively, consistent with reports of a variety of dynamic moduli, their dependencies on the rate and amplitude of load application, and some features associated with preconditioning.
5904418
Dynamic properties of lung parenchyma: mechanical contributions of fiber network and interstitial cells.
We investigated the contributions of the connective tissue fiber network and interstitial cells to parenchymal mechanics in a surfactant-free system. In eight strips of uniform dimension from guinea pig lung, we assessed the storage (G') and loss (G") moduli by using pseudo-random length oscillations containing a specially designed set of seven frequencies from 0.07 to 2.4 Hz at baseline, during methacholine (MCh) challenge, and after death of the interstitial cells. Measurements were made at mean forces of 0.5 and 1 g and strain amplitudes of 5, 10, and 15% and were repeated 12 h later in the same, but nonviable samples. The results were interpreted using a linear viscoelastic model incorporating both tissue damping (G) and stiffness (H). The G' and G" increased linearly with the logarithm of frequency, and both G and H showed negative strain amplitude and positive mean force dependence. After MCh challenge, the G' and G" spectra were elevated uniformly, and G and H increased by < 15%. Tissue stiffness, strain amplitude, and mean force dependence were virtually identical in the viable and nonviable samples. The G and hence energy dissipation were approximately 10% smaller in the nonviable samples due to absence of actin-myosin cross-bridge cycling. We conclude that the connective tissue network may also dominate parenchymal mechanics in the intact lung, which can be influenced by the tone or contraction of interstitial cells.
22238941
Extracellular matrix proteins: a positive feedback loop in lung fibrosis?
Lung fibrosis is characterized by excessive deposition of extracellular matrix. This not only affects tissue architecture and function, but it also influences fibroblast behavior and thus disease progression. Here we describe the expression of elastin, type V collagen and tenascin C during the development of bleomycin-induced lung fibrosis. We further report in vitro experiments clarifying both the effect of myofibroblast differentiation on this expression and the effect of extracellular elastin on myofibroblast differentiation. Lung fibrosis was induced in female C57Bl/6 mice by bleomycin instillation. Animals were sacrificed at zero to five weeks after fibrosis induction. Collagen synthesized during the week prior to sacrifice was labeled with deuterium. After sacrifice, lung tissue was collected for determination of new collagen formation, microarray analysis, and histology. Human lung fibroblasts were grown on tissue culture plastic or BioFlex culture plates coated with type I collagen or elastin, and stimulated to undergo myofibroblast differentiation by 0-10 ng/ml transforming growth factor (TGF)β1. mRNA expression was analyzed by quantitative real-time PCR. New collagen formation during bleomycin-induced fibrosis was highly correlated to gene expression of elastin, type V collagen and tenascin C. At the protein level, elastin, type V collagen and tenascin C were highly expressed in fibrotic areas as seen in histological sections of the lung. Type V collagen and tenascin C were transiently increased. Human lung fibroblasts stimulated with TGFβ1 strongly increased gene expression of elastin, type V collagen and tenascin C. The extracellular presence of elastin increased gene expression of the myofibroblastic markers α smooth muscle actin and type I collagen. The extracellular matrix composition changes dramatically during the development of lung fibrosis. The increased levels of elastin, type V collagen and tenascin C are probably the result of increased expression by fibroblastic cells; reversely, elastin influences myofibroblast differentiation. This suggests a reciprocal interaction between fibroblasts and the extracellular matrix composition that could enhance the development of lung fibrosis.
38276700
Elastin in lung development and disease.
Elastic fibres are present in lung structures including alveoli, alveolar ducts, airways, vasculature and pleura. The rate of lung elastin synthesis is greatest during fetal and neonatal development, and is minimal in the healthy adult. We have determined that glucocorticoids up-regulate fetal lung tropoelastin expression while concomitantly accelerating terminal airspace maturation. Because there is minimal turnover of elastin in healthy adult lung, the elastin incorporated into the lung early in development supports lung function for the normal lifespan. However, in the adult lung, in pathological circumstances such as emphysema or pulmonary fibrosis there may be reactivation of elastin expression. We have found in silica-induced pulmonary fibrosis that expression of tropoelastin is primarily increased in the walls and the septal tips of the alveolus, with modest increases in other compartments which normally express tropoelastin during development. This finding suggests that the mesenchymal cell of the alveolar wall increases tropoelastin expression during fibrotic disorders. In emphysema and fibrosis, elastin is present in abnormal-appearing, probably non-functional, elastic fibres, suggesting that the adult lung cannot recapitulate the elastic fibre assembly mechanisms operative during normal lung growth.
23726982
Studies on the chemical nature of elastin fluorescence.
Two fluorescent fractions were found in total acid hydrolysate of elastin. The fraction with higher chromatography mobility in isopropyl alcohol/conc. ammonia/water (9 : 1 : 2) was purified by multiple preparative paper chromatography in the same solvent system, and by gel chromatography on Sephadex G-25, ion-exchange chromatography on phosphocellulose and another gel chromatography on Sephadex G-10. The purified material was chromatographically homogeneous, had an ultraviolet absorption maximum at 315 nm and exhibited a strong 320/405 nm fluorescence. 1H- and 13C-NMR spectra were in good agreement with those published previously [6] for pyridinoline, a lysine derived fluorescent compound in collagen. The major part of the fluorescent material present in acid hydrolysate of elastin was always contaminated, even after complex purification procedures. It is concluded that elastin contains several fluorophores, one of which is a cross-linking tricarboxylic amino acid with a pyridinium ring having very probably the structure of 3-(2-amino-2-carboxyethyl)-1-(5-amino-5-carboxy-2-hydroxy-pentyl)-4-(3-amino-3- carboxypropyl)-5-hydroxypyridinium. The position of 2-amino-2-carboxyethyl and 3-amino-3-carboxypropyl residues has not been definitely established and can be interchanged.
13991442
Desmosine as a biomarker of elastin degradation in COPD: current status and future directions
Desmosine (DES) and isodesmosine (IDES) are two unusual, tetrafunctional, pyridinium ring-containing amino acids involved in elastin cross-linking. Being amino acids unique to mature, cross-linked elastin, they are useful for discriminating peptides derived from elastin breakdown from precursor elastin peptides. According to these features, DES and IDES have been extensively discussed as potentially attractive indicators of elevated lung elastic fibre turnover and markers of the effectiveness of agents with the potential to reduce elastin breakdown. In the present manuscript, immunology-based and separation methods for the evaluation of DES and IDES are discussed, along with studies reporting increased levels of urine excretion in chronic obstructive pulmonary disease (COPD) patients with and without α1-antitrypsin deficiency. The results of the application of DES and IDES as surrogate end-points in early clinical trials in COPD are also reported. Finally, recent advances in detection techniques, including liquid chromatography tandem mass spectrometry and high-performance capillary electrophoresis with laser-induced fluorescence, are discussed. These techniques allow detection of DES and IDES at very low concentration in body fluids other than urine, such as plasma or sputum, and will help the understanding of whether DES and IDES are potentially useful in monitoring therapeutic intervention in COPD.
43353112
Extracellular matrix remodeling of lung alveolar walls in three dimensional space identified using second harmonic generation and multiphoton excitation fluorescence.
The structural reorganization of extracellular matrix (ECM) is an important feature of peripheral lung tissue remodeling in chronic obstructive pulmonary disease (COPD). Ordered ECM macromolecules such as the fibril-forming collagens produce second harmonic generation (SHG) signal without the need for any exogenous label, while ECM macromolecules such as the elastin fibers generate MPEF signal due to their endogenous fluorescence characteristics. Both these signals can be captured simultaneously to provide spatially resolved 3D structural reorganization of ECM matrix. In this study, SHG and MPEF microscopy methods were used to examine structural remodeling of the ECM matrix in human lung alveolar walls undergoing severe emphysematous destruction. Flash frozen lung samples removed from two patients undergoing lung transplantation for severe COPD (n=4) were compared to similar samples from an unused donor lung (n=2) that served as a control. The imaging operations were performed directly on these tissue sections at least three different areas. The generated spatially resolved 3D images showed the distribution of collagen and elastin in the alveolar walls. In the case of the control, we found well ordered alveolar walls with a composite type structure made up of collagen bands and relatively fine elastic fibers. In contrast, lung tissues undergoing emphysematous destruction were highly disorganized with significantly increased alveolar wall thickness compared to the control. We conclude that these non-invasive imaging modalities provide spatially resolved 3D images with spectral specificities that are sensitive enough to identity the ECM structural changes associated with emphysematous destruction.
37919936
Minimally invasive imaging method based on second harmonic generation and multiphoton excitation fluorescence in translational respiratory research
For translational respiratory research including in the development of clinical diagnostic tools, a minimally invasive imaging method, which can provide both cellular and extracellular structural details with sufficient specificity, sensitivity and spatial resolution, is particularly useful. Multiphoton microscopy causes excitation of endogenously fluorescent macromolecular systems and induces highly specific second harmonic generation signals from non‐centrosymmetric macromolecules such as fibrillar collagens. Both these signals can be captured simultaneously to provide spatially resolved 3D structural organization of extracellular matrix as well as the cellular morphologies in their native states. Besides briefly discussing the fundamentals of multiphoton excitation fluorescence and harmonic generation signals and the instrumentation details, this review focuses on the specific applications of these imaging modalities in lung structural imaging, particularly morphological features of alveolar structures, visualizing and quantifying extracellular matrix remodelling accompanying emphysematous destructions as well as the IPF, detecting lung cancers and the potential use in the tissue engineering applications.
25833460
Optical diagnostics of tissue pathology by multiphoton microscopy.
IMPORTANCE OF THE FIELD
Advances in nonlinear optical microscopy have led to new imaging modalities that can be used for diagnosing pathological tissues. Intrinsic optical sectioning, reduced photodamage and greater specimen penetration depth allow three-dimensional, high-contrast, tissue imaging to be performed with multiphoton techniques. Although autofluorescence can be useful in imaging cellular architecture, it is the polarization-based technique of second harmonic generation (SHG) that is effective in visualizing non-centrosymmetric biological structures such as collagen and muscle fibers without extrinsic labeling. In addition, third harmonic generation (THG) offers another contrast mechanism for imaging the morphology of tissue constituents. These techniques offer the possibility of diagnosing normal and diseased states in selected epithelial tissues, often without the introduction of extrinsic labels.


AREAS COVERED IN THIS REVIEW
In this review, the physical principles of multiphoton fluorescence excitation, SHG and THG processes are discussed. In addition, the basic instrument set up of a multiphoton microscope along with recent advances in multiphoton microscopy instrumentation are presented. Examples from imaging tissue pathologies from organs such as skin, cornea, lung and the liver are illustrated. Finally, developments leading to miniaturized or endoscopic instrumentation that allows real-time diagnosis of tissue pathologies in accessible epithelial tissues are discussed.


WHAT THE READER WILL GAIN
From this review, the reader will gain knowledge of the basic principles of multiphoton microscopy and the technical information of building a multiphoton instrument. In addition, the reader will appreciate the ability of this technique in the diagnostics of pathological tissues and gain insights into the future development of this technique.


TAKE HOME MESSAGE
Intrinsic fluorescence and harmonic generation signals are capable of differentiating normal and pathological epithelial tissues. Further technical advances leading to improved imaging depths, multimodal imaging capabilities and endoscopic instrumentation may enable the effective diagnostics of selected epithelial tissues in the clinical setting.
24235816
Three‐dimensional investigation and scoring of extracellular matrix remodeling during lung fibrosis using multiphoton microscopy
The organization of collagen during fibrotic processes is poorly characterized because of the lack of appropriate methodologies. Here we show that multimodal multiphoton microscopy provides novel insights into lung fibrosis. We characterize normal and fibrotic pulmonary tissue in the bleomycin model, and show that second‐harmonic generation by fibrillar collagen reveals the micrometer‐scale three‐dimensional spatial distribution of the fibrosis. We find that combined two‐photon excited fluorescence and second‐harmonic imaging of unstained lung tissue allows separating the inflammatory and fibrotic steps in this pathology, underlining characteristic features of fibroblastic foci in human Idiopathic Pulmonary Fibrosis samples. Finally, we propose phenomenological scores of lung fibrosis and we show that they unambiguously sort out control and treated mice, with a better sensitivity and reproducibility in the subpleural region. These results should be readily generalized to other organs, as an accurate method to assess extracellular matrix remodeling during fibrosis. Microsc. Res. Tech., 2007. © 2006 Wiley‐Liss, Inc.
4669290
Measurement of MMP-9 and -12 degraded elastin (ELM) provides unique information on lung tissue degradation
BackgroundElastin is an essential component of selected connective tissues that provides a unique physiological elasticity. Elastin may be considered a signature protein of lungs where matrix metalloprotease (MMP) -9-and -12, may be considered the signature proteases of the macrophages, which in part are responsible for tissue damage during disease progression. Thus, we hypothesized that a MMP-9/-12 generated fragment of elastin may be a relevant biochemical maker for lung diseases.MethodsElastin fragments were identified by mass-spectrometry and one sequence, generated by MMP-9 and -12 (ELN-441), was selected for monoclonal antibody generation and used in the development of an ELISA. Soluble and insoluble elastin from lung was cleaved in vitro and the time-dependent release of fragments was assessed in the ELN-441 assay. The release of ELN-441 in human serum from patients with chronic obstructive pulmonary disease (COPD) (n = 10) and idiopathic pulmonary fibrosis (IPF) (n = 29) were compared to healthy matched controls (n = 11).ResultsThe sequence ELN-441 was exclusively generated by MMP-9 and -12 and was time-dependently released from soluble lung elastin. ELN-441 levels were 287% higher in patients diagnosed with COPD (p < 0.001) and 124% higher in IPF patients (p < 0.0001) compared with controls. ELN-441 had better diagnostic value in COPD patients (AUC 97%, p = 0.001) than in IPF patients (AUC 90%, p = 0.0001). The odds ratios for differentiating controls from COPD or IPF were 24 [2.06–280] for COPD and 50 [2.64–934] for IPF.ConclusionsMMP-9 and -12 time-dependently released the ELN-441 epitope from elastin. This fragment was elevated in serum from patients with the lung diseases IPF and COPD, however these data needs to be validated in larger clinical settings.
25630989
Amount of elastic fibers predicts prognosis of idiopathic pulmonary fibrosis.
BACKGROUND
Elastic fibers enhance the stiffness of fibrotic tissues, but their role in the pathophysiology of idiopathic pulmonary fibrosis (IPF) has not been fully examined. The aim of this study was to determine clinical significance of the amount of elastic fibers in IPF.


METHODS
We studied the surgical lung biopsy specimens of 43 patients with IPF. Histological specimens were stained using the Elastica Van Gieson method and digital images were taken. The number of pixels containing elastic fibers was divided by the number occupied by fibrotic tissue, from which the proportion of elastic fibers (elastic fiber score, %) was calculated. The relationships between the elastic fiber score and clinical, radiological and pathological findings, and prognosis were explored.


RESULTS
The median elastic fiber score was 10.9% (range 5.1-23.3%). Scores were inversely correlated with % predicted forced vital capacity (r -0.451, p-value 0.003) and positively correlated with decline in forced vital capacity over 12 months (r -0.475, p-value 0.033). Furthermore, elastic fiber score correlated with the extent of fibrotic lesions assessed on high resolution computed tomography as well as the degree of collagen deposition on biopsy specimens. Patients with high elastic fiber scores had significantly worse outcomes than those with low scores (5-year survival rate was 48.7% and 84.0%, respectively, p-value 0.024), and elastic fiber score was an independent predictor of poor prognosis (hazard ratio 1.21, p-value 0.005).


CONCLUSION
The amount of elastic fiber in fibrotic tissue is a prognostic indicator in patients with IPF.
29958401
Elastin gene expression is upregulated during pulmonary fibrosis.
Elastin is a chief component of lung interstitium, and it is central to lung morphology and function. Efforts to understand the pathogenesis of pulmonary fibrosis have focused primarily upon collagen turnover in the lung; few studies have focused on elastin. In this study, we examined steady-state elastin mRNA levels after lung injury in the mouse induced by (1) butylated hydroxytoluene (BHT) which causes acute lung injury with recovery, (2) BHT + 70% O2 (fibrosis), or (3) 70% O2. Total lung elastin mRNA increased 70-80-fold on d10-14 after BHT/O2, but was unchanged after BHT or O2 alone. In situ hybridization studies localized elastin mRNA to cells in the muscularis of conducting airways and to scattered interstitial cells in fibrotic foci. Elastic fiber morphology was markedly distorted after BHT/O2. Thus, marked upregulation of elastin gene expression is correlated with the histopathology of fibrotic lung disease.
797822
Elastin and collagen degradation products in urine of patients with cystic fibrosis.
Elastin degradation has been reported to be increased in patients with cystic fibrosis (CF). In order to further explore evidence for elastin degradation in a group of 18 patients with CF with a wide range of disease severity, we used an isotope dilution method to measure urinary desmosine (DES) and isodesmosine (IDES), amino acids derived exclusively from cross-linked elastin, and hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP), amino acids derived exclusively from cross-linked collagen. Urinary DES and IDES (mean +/- SD) were 23.9 +/- 30.7 and 18.5 +/- 22.4 micrograms/g creatinine, respectively, in the patients with CF versus 7.5 +/- 1.7 and 6.8 +/- 1.4 micrograms/g creatinine, respectively, in 10 healthy control subjects (p < 0.001); only two patients with CF had DES values within the control range. The values of urinary HP and LP in the CF group were 54.9 +/- 39.1 and 12.3 +/- 8.6 nmol/mmol creatinine, respectively, versus 24.5 +/- 5.8 and 5.1 +/- 2.7 nmol/mmol creatinine, respectively, in the controls (p < 0.005). Both HP and LP were highly correlated (r = 0.71, p < 0.0001). Patients with CF had active pulmonary inflammation; neutrophils were abundant in the bronchoalveolar lavage fluid of the CF group and correlated with elastase activity measured with methoxysuccinyl Ala-Ala-Pro-Val paranitroanilide (r = 0.61, p < 0.05). Airway neutrophils had decreased expression of the complement receptor CR1 (CR1/CR3 of 0.17 +/- 0.15 versus 1.0 for blood neutrophils), a change known to be caused by uninhibited neutrophil elastase. We conclude that lung elastin is the most likely source of the increased DES and IDES in CF.