Distinctive roles of fibrillar collagen I and collagen III in mediating fibroblast-matrix interaction: A nanoscopic study.

One major goal in tissue engineering is to create functional materials, mimicking scaffolds in native tissues, to modulate cell function for tissue repair. Collagen is the most abundant structural protein in human body. Though collagen I (COLI) and collagen III (COLIII) are the predominant collagen types in connective tissues and they form stable hybrid fibrils at varied ratios, cell responses to the hybrid matrices are underinvestigated. In this work, we aim to explicate the distinctive roles of COLI and COLIII in fibroblast activation. Unidirectionally aligned COLI, COLIII and COLI-COLIII hybrid nanofibrils were generated via epitaxial growth of collagen on mica. AFM analyses revealed that, with the increase of COLI/COLIII ratio, the fibril width and stiffness increased and the binding affinity of cells to the matrix decreased. A hybrid matrix was found to activate fibroblasts the most effectively, characterized by extensive cell polarization with rigid stress fiber bundles and high α-SMA expression, and by the highest-level of collagen synthesis. It is ascribed to the fine balance between biochemical and biophysical cues achieved on the hybrid matrix. Thus, matrices of aligned COLI-COLIII hybrid fibrils and their derived multifunctional composites can be good candidates of implantation scaffolds for tissue regeneration.

In vitro fibrillogenesis of tropocollagen type III in collagen type I affects its relative fibrillar topology and mechanics.

Tropocollagen types I and III were simultaneously fibrilized in vitro, and the differences between the geometric and mechanical properties of the heterotypic fibrils with different mixing ratios of tropocollagen III to I were investigated. Transmission electron microscopy was used to confirm the simultaneous presence of both tropocollagen types within the heterotypic fibrils. The incorporation of collagen III in I caused the fibrils to be thinner with a shorter D-banding than pure collagen I. Hertzian contact model was used to obtain the elastic moduli from atomic force microscope indentation testing using a force volume analysis. The results indicated that an increase in the percentage of tropocollagen III reduced the mechanical stiffness of the obtained fibrils. The mechanical stiffness of the collagen fibrils was found to be greater at higher loading frequencies. This observation might explain the dominance of collagen III over I in soft distensible organs such as human vocal folds.

Wavelength-Dependent Second Harmonic Generation Circular Dichroism for Differentiation of Col I and Col III Isoforms in Stromal Models of Ovarian Cancer Based on Intrinsic Chirality Differences.

Extensive remodeling of the extracellular matrix (ECM) occurs in many epithelial cancers. For example, in ovarian cancer, upregulation of collagen isoform type III has been linked to invasive forms of the disease, and this change may be a potential biomarker. To examine this possibility, we implemented wavelength-dependent second harmonic generation circular dichroism (SHG-CD) imaging microscopy to quantitatively determine changes in chirality in ECM models comprised of different Col I/Col III composition. In these models, Col III was varied between 0 and 40%, and we found increasing Col III results in reduced net chirality, consistent with structural biology studies of Col I and III in tissues where the isoforms comingle in the same fibrils. We further examined the wavelength dependence of the SHG-CD to both optimize the response and gain insight into the underlying mechanism. We found using shorter SHG excitation wavelengths resulted in increased SHG-CD sensitivity, where this is consistent with the electric-dipole-coupled oscillator model suggested previously for the nonlinear chirality response from thin films. Moreover, the sensitivity is further consistent with the wavelength dependency of SHG intensity fit to a two-state model of the two-photon absorption in collagen. We also provide experimental calibration protocols to implement the SHG-CD modality on a laser scanning microscope. We last suggest that the technique has broad applicability in probing a wide range of diseased states with changes in collagen molecular structure.

Collagen type III glomerulopathy: case report and review of the literature
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To summarize the clinical and pathological features of collagen type III glomerulopathy, the present report describes a case of collagen type III glomerulopathy and reviewed more than 60 cases recorded by other groups in English literature over the last few years. A 24-year-old female patient was admitted because of lower limbs edema, without any other discomforts. The lab examination of the patient reported proteinuria (2.42 g/24 h urine), microscopic hematuria, and the serum creatinine was 71 µmol/L. She received renal biopsy. The immunofluorescence staining results showed that collagen type III expression was positive. The electron microscopy examination showed that the mesangial regions were widened, with visible fine fibers in it. The periodic stripes of collagen fibers could be seen on some fine fiber-like substance under a high-magnification microscope, the diameters of the fiber-like substances were 40 - 70 nm. Serum collagen type III N-peptide (PIIIP N-P) was 97.94 ng/mL. After the patient received benazepril 10 mg per day and symptomatic treatments (Chinese drug, Cordyceps Capsules 0.5 g per day), her proteinuria resolved (~ 0.5 g/24 hour urine).

The structural and optical properties of type III human collagen biosynthetic corneal substitutes.

The structural and optical properties of clinically biocompatible, cell-free hydrogels comprised of synthetically cross-linked and moulded recombinant human collagen type III (RHCIII) with and without the incorporation of 2-methacryloyloxyethyl phosphorylcholine (MPC) were assessed using transmission electron microscopy (TEM), X-ray scattering, spectroscopy and refractometry. These findings were examined alongside similarly obtained data from 21 human donor corneas. TEM demonstrated the presence of loosely bundled aggregates of fine collagen filaments within both RHCIII and RHCIII-MPC implants, which X-ray scattering showed to lack D-banding and be preferentially aligned in a uniaxial orientation throughout. This arrangement differs from the predominantly biaxial alignment of collagen fibrils that exists in the human cornea. By virtue of their high water content (90%), very fine collagen filaments (2-9 nm) and lack of cells, the collagen hydrogels were found to transmit almost all incident light in the visible spectrum. They also transmitted a large proportion of UV light compared to the cornea which acts as an effective UV filter. Patients implanted with these hydrogels should be cautious about UV exposure prior to regrowth of the epithelium and in-growth of corneal cells into the implants.

Clinical and morphological features of collagen type III glomerulopathy: a report of nine cases from a single institution.

AIMS: We report nine Chinese patients with collagen type III glomerulopathy. METHODS AND RESULTS: Two males and seven females were studied, ranging in age from 21 to 67 years. Proteinuria and hypertension were the most common symptoms, with incidences of 88.9 and 77.8%, respectively. Two patients had abnormal renal function. Their histological appearances varied. Massive eosinophilic and weakly periodic acid-Schiff (PAS)-positive substances were deposited along the capillary loops and in the mesangial area in three cases, while others had thickened capillary walls with a chain-like structure or double-contour appearance of the PAS- and silver-stained sections. Immunofluorescence analysis showed the abundant deposition of collagen type III. Electron microscopy revealed massive scattered or bundle-shaped fibre-like materials in the subendothelial and mesangial areas. During follow-up, 44.4% of the patients suffered a doubling of serum creatinine. The level of serum creatinine at biopsy was an independent predictor of this doubled serum creatinine value. CONCLUSIONS: Collagen type III deposits in the subendothelial and mesangial areas. Some patients show global nodular lesions, while others show subtle changes only via PAS/silver staining. Proteinuria and hypertension are the most common symptoms, and the serum creatinine level at biopsy is an independent predictor of the doubling of serum creatinine during follow-up.

3-D ultrastructure and collagen composition of healthy and overloaded human tendon: evidence of tenocyte and matrix buckling.

Achilles tendinopathies display focal tissue thickening with pain and ultrasonography changes. Whilst complete rupture might be expected to induce changes in tissue organization and protein composition, little is known about the consequences of non-rupture-associated tendinopathies, especially with regards to changes in the content of collagen type I and III (the major collagens in tendon), and changes in tendon fibroblast (tenocyte) shape and organization of the extracellular matrix (ECM). To gain new insights, we took biopsies from the tendinopathic region and flanking healthy region of Achilles tendons of six individuals with clinically diagnosed tendinopathy who had no evidence of cholesterol, uric acid and amyloid accumulation. Biochemical analyses of collagen III/I ratio were performed on all six individuals, and electron microscope analysis using transmission electron microscopy and serial block face-scanning electron microscopy were made on two individuals. In the tendinopathic regions, compared with the flanking healthy tissue, we observed: (i) an increase in the ratio of collagen III : I proteins; (ii) buckling of the collagen fascicles in the ECM; (iii) buckling of tenocytes and their nuclei; and (iv) an increase in the ratio of small-diameter : large-diameter collagen fibrils. In summary, load-induced non-rupture tendinopathy in humans is associated with localized biochemical changes, a shift from large- to small-diameter fibrils, buckling of the tendon ECM, and buckling of the cells and their nuclei.

A quantitative comparison of morphological and histological characteristics of collagen in the rabbit medial collateral ligament.

Collagen fiber is one of the critical factors in determining mechanical properties of ligaments and both the morphological and histological characteristics of collagen have been widely studied. However, there was still no consensus about whether the morphological characteristics of collagen correlated with its histological characteristics in physiological ligaments. Rabbit medial collateral ligaments (MCLs) were measured under a transmission electron microscope and a polarized light microscope plus picrosirius red-staining to obtain the distributions of collagen fibril diameters and types at different anatomical sites of rabbit MCLs, respectively. The correlation between the fibril diameter and type was determined by a correlation analysis. The collagen fibril diameters at the different anatomical sites had different distributions (unimodal or bimodal) and mean fibril diameters were found to increase significantly from the anterior part to the posterior part (P=0.0482) as well as from the proximal to the distal sections (P=0.0208). Type I collagen in the core portion of MCLs was significantly less than at the other four peripheral areas (P<0.005) but no significant variation was found in each respective portion (P>0.05). The low coefficient in the correlation analysis (r=0.3759) demonstrated collagen fibril diameters had no correlation with collagen types. This may provide a new view of collagen types in studying the mechanical behavior of ligaments.

[Jejunum intersticium in mongolian gerbils after the flight on spacecraft Foton-M3].

Methods of light optical microscopy were used to explore histoarchitectonics, topography and tinctorial properties of the extracellular phase of fibers of jejunum wall intersticium in Mongolian gerbils following 12-day orbital flight aboard Foton-M3, ground-based simulation of the spaceflight factors in the KONTUR-L facility, and in the group of biological control Postflight destructive changes were found in reticulin fibers (type-III collagen) of villi stroma, intercrypt intersticium and submucosa. Local acidophilia and fiber homogenization formed in type I collagen present in the intestinal subserous layer, muscular layers endomysium and submucose against the background of progressing edema and arterial, venous plethora and lymphostasis. Elastic component of the intersticium was disarranged in the structures of internal elastic membrane of submucous vessels, fragmented and partly reduced. Simulation of the orbital factors, except for microgravity, in the KONTUR-L facility called forth similar, although less often and diffuse, changes in intersticium fibers. The results of examination of intestinal intersticium fibers in the vivarium control gerbils discovered expressed species characters that should be taken into account by investigators, especially when comparing with data obtained from other animal species.

Ultrastructural scoring of skin biopsies for diagnosis of vascular Ehlers-Danlos syndrome.

Vascular Ehlers-Danlos syndrome (vEDS) results from a mutation in the gene encoding alpha-1, type III pro-collagen (COL3A1) and confers fragility to skin, ligament and vascular tissue. We tested the value of skin biopsy for diagnosis of vEDS through an ultrastructure scoring procedure. Study design was a multicentric, case-control, blinded trial consisting of two phases: phase 1 was to identify an ultra-structure score providing the best discriminative value for vEDS and phase 2 was to replicate this result in a different population. We enrolled 103 patients, 66 cases defined through the revised nosology for Ehlers-Danlos syndromes and 37 control subjects selected from patients referred for other pathologies. Ultrastructure of extracellular matrix was read by three to five experienced pathologists blinded for diagnosis. We used the receiver operating curves and logistic regression analysis for ranking ultrastructure scores. We created a detailed description of lesions observed in vEDS patients with 27 items (coded 0 or 1). In the phase 1 (17 cases and 20 controls), abnormal fibroblast shape, presence of lysosomes in the fibroblast and abnormal basal lamina were found to be independent discriminative items. Addition of these three items (defining an ultrastructure score) had the best diagnosis value (area under the curve (AUC) = 0.96). In the phase 2 (49 cases, 17 controls), ultrastructure score provided odds ratio of 9.76 (95 % CI 2.91-32.78), and AUC of 0.90. The ultrastructure score of skin biopsy has predictive value for the diagnosis of vEDS. Presence of two or more signs (either abnormal fibroblast, presence of lysosomes in the fibroblast or abnormal basal lamina) is very evocative of vEDS.

Getting to the heart of cardiac remodeling; how collagen subtypes may contribute to phenotype.

The objective of this study was to investigate the nature and biomechanical properties of collagen fibers within the human myocardium. Targeting cardiac interstitial abnormalities will likely become a major focus of future preventative strategies with regard to the management of cardiac dysfunction. Current knowledge regarding the component structures of myocardial collagen networks is limited, further delineation of which will require application of more innovative technologies. We applied a novel methodology involving combined confocal laser scanning and atomic force microscopy to investigate myocardial collagen within ex-vivo right atrial tissue from 10 patients undergoing elective coronary bypass surgery. Immuno-fluorescent co-staining revealed discrete collagen I and III fibers. During single fiber deformation, overall median values of stiffness recorded in collagen III were 37±16% lower than in collagen I [p<0.001]. On fiber retraction, collagen I exhibited greater degrees of elastic recoil [p<0.001; relative percentage increase in elastic recoil 7±3%] and less energy dissipation than collagen III [p<0.001; relative percentage increase in work recovered 7±2%]. In atrial biopsies taken from patients in permanent atrial fibrillation (n=5) versus sinus rhythm (n=5), stiffness of both collagen fiber subtypes was augmented (p<0.008). Myocardial fibrillar collagen fibers organize in a discrete manner and possess distinct biomechanical differences; specifically, collagen I fibers exhibit relatively higher stiffness, contrasting with higher susceptibility to plastic deformation and less energy efficiency on deformation with collagen III fibers. Augmented stiffness of both collagen fiber subtypes in tissue samples from patients with atrial fibrillation compared to those in sinus rhythm are consistent with recent published findings of increased collagen cross-linking in this setting.

Normal skin and hypertrophic scar fibroblasts differentially regulate collagen and fibronectin expression as well as mitochondrial membrane potential in response to basic fibroblast growth factor

Basic fibroblast growth factor (bFGF) regulates skin wound healing; however, the underlying mechanism remains to be defined. In the present study, we determined the effects of bFGF on the regulation of cell growth as well as collagen and fibronectin expression in fibroblasts from normal human skin and from hypertrophic scars. We then explored the involvement of mitochondria in mediating bFGF-inducedeffects on the fibroblasts. We isolated and cultivated normal and hypertrophic scar fibroblasts from tissue biopsies of patients who underwent plastic surgery for repairing hypertrophic scars. The fibroblasts were then treated with different concentrations of bFGF (ranging from 0.1 to 1000 ng/mL). The growth of hypertrophic scar fibroblasts became slower with selective inhibition of type I collagen production after exposure to bFGF. However, type III collagen expression was affected in both normal and hypertrophic scar fibroblasts. Moreover, fibronectin expression in the normal fibroblasts was up-regulated after bFGF treatment. bFGF (1000 ng/mL) also induced mitochondrial depolarization in hypertrophic scar fibroblasts (P < 0.01). The cellular ATP level decreased in hypertrophic scar fibroblasts (P < 0.05), while it increased in the normal fibroblasts following treatment with bFGF (P < 0.01). These data suggest that bFGF has differential effects and mechanisms on fibroblasts of the normal skin and hypertrophic scars, indicating that bFGF may play a role in the early phase of skin wound healing and post-burn scar formation.

Normal skin and hypertrophic scar fibroblasts differentially regulate collagen and fibronectin expression as well as mitochondrial membrane potential in response to basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) regulates skin wound healing; however, the underlying mechanism remains to be defined. In the present study, we determined the effects of bFGF on the regulation of cell growth as well as collagen and fibronectin expression in fibroblasts from normal human skin and from hypertrophic scars. We then explored the involvement of mitochondria in mediating bFGF-induced effects on the fibroblasts. We isolated and cultivated normal and hypertrophic scar fibroblasts from tissue biopsies of patients who underwent plastic surgery for repairing hypertrophic scars. The fibroblasts were then treated with different concentrations of bFGF (ranging from 0.1 to 1000 ng/mL). The growth of hypertrophic scar fibroblasts became slower with selective inhibition of type I collagen production after exposure to bFGF. However, type III collagen expression was affected in both normal and hypertrophic scar fibroblasts. Moreover, fibronectin expression in the normal fibroblasts was up-regulated after bFGF treatment. bFGF (1000 ng/mL) also induced mitochondrial depolarization in hypertrophic scar fibroblasts (P < 0.01). The cellular ATP level decreased in hypertrophic scar fibroblasts (P < 0.05), while it increased in the normal fibroblasts following treatment with bFGF (P < 0.01). These data suggest that bFGF has differential effects and mechanisms on fibroblasts of the normal skin and hypertrophic scars, indicating that bFGF may play a role in the early phase of skin wound healing and post-burn scar formation.

Connective tissue alteration in abdominal wall hernia.

BACKGROUND: The aetiology and pathogenesis of abdominal wall hernia formation is complex. Optimal treatment of hernias depends on a full understanding of the pathophysiological mechanisms involved in their formation. The aim of this study was to review the literature on specific collagen alterations in abdominal wall hernia formation. METHODS: A computer-assisted search of the medical databases PubMed and Embase was performed, together with a cross-reference search of eligible papers. RESULTS: Fifty-two papers were included. Collagen alteration depended on the type of hernia; there were more pronounced changes in patients with a direct inguinal hernia than in those with an indirect inguinal hernia, recurrent inguinal hernia or incisional hernia. A consistent finding was a significant increase in immature type III collagen relative to the stronger type I collagen in patients with a hernia. This resulted in thinner collagen fibres with a correspondingly diminished biomechanical strength. It has been suggested that these alterations are due to variation in the synthesis, maturation or degradation of collagen by matrix metalloproteinases, in combination or alone. CONCLUSION: Hernia formation and recurrence is associated with altered collagen metabolism manifested by a decreased type I:III collagen ratio.

Morphological and biochemical analysis of intact and opaque cornea in dogs.

The arrangement of collagen fibrils and glycosaminoglycans (GAGs) in substantia propria are important for maintaining transparency of the cornea. Interferences in collagen fibrils and GAG production could be adversative to corneal integrity. In this study, six dogs consisting of four Beagles with normal cornea (normal), one Beagles with opaque cornea (sample No. 1) and one Shih Tzu with neovascularization opaque cornea (sample No.2) were used. All samples were observed morphologically by light and electron microscopes to obtain diameter and distribution of collagen fibrils in substantia propria and were performed biochemically to investigate into GAGs and collagen types. The average diameter of collagen fibrils in the intact cornea of normal, sample No.1 and No.2 was 33.2, 35.0 and 25.0 nm, respectively. The percentage of matrix per unit area was 67% in normal, 87% in sample No.1 and 28.3% in sample No.2. The type III collagen ratio was 25.3% in normal, 21.3% in sample No.1 and 35.8% in sample No.2. The relative amount of heparan sulfate, chondroitin sulfate, dermatan sulfate and keratin sulfate was 1.5, 9.7, 51.1 and 37.7% in normal, 3.3, 26.0, 45.7 and 23.7% in sample No.1 and 1.2, 18.0, 16.6 and 54.1% in sample No.2. Hyaluronic acid was found only in sample No.1 with a relative amount of 1.3%. Since there was some relationship between collagen formation and GAGs composition, it might be speculated that disturbance in arrangement of collagen fibrils and GAG metabolism especially in substantia propria would bring up opacity of the cornea.

Hemizygous deletion of COL3A1, COL5A2, and MSTN causes a complex phenotype with aortic dissection: a lesson for and from true haploinsufficiency.

Aortic dilatation/dissection (AD) can occur spontaneously or in association with genetic syndromes, such as Marfan syndrome (MFS; caused by FBN1 mutations), MFS type 2 and Loeys-Dietz syndrome (associated with TGFBR1/TGFBR2 mutations), and Ehlers-Danlos syndrome (EDS) vascular type (caused by COL3A1 mutations). Although mutations in FBN1 and TGFBR1/TGFBR2 account for the majority of AD cases referred to us for molecular genetic testing, we have obtained negative results for these genes in a large cohort of AD patients, suggesting the involvement of additional genes or acquired factors. In this study we assessed the effect of COL3A1 deletions/duplications in this cohort. Multiplex ligation-dependent probe amplification (MLPA) analysis of 100 unrelated patients identified one hemizygous deletion of the entire COL3A1 gene. Subsequent microarray analyses and sequencing of breakpoints revealed the deletion size of 3,408,306 bp at 2q32.1q32.3. This deletion affects not only COL3A1 but also 21 other known genes (GULP1, DIRC1, COL5A2, WDR75, SLC40A1, ASNSD1, ANKAR, OSGEPL1, ORMDL1, LOC100129592, PMS1, MSTN, C2orf88, HIBCH, INPP1, MFSD6, TMEM194B, NAB1, GLS, STAT1, and STAT4), mutations in three of which (COL5A2, SLC40A1, and MSTN) have also been associated with an autosomal dominant disorder (EDS classical type, hemochromatosis type 4, and muscle hypertrophy). Physical and laboratory examinations revealed that true haploinsufficiency of COL3A1, COL5A2, and MSTN, but not that of SLC40A1, leads to a clinical phenotype. Our data not only emphasize the impact/role of COL3A1 in AD patients but also extend the molecular etiology of several disorders by providing hitherto unreported evidence for true haploinsufficiency of the underlying gene.

Lamina propria cellularity and collagen composition: an integrated assessment of structure in humans.

OBJECTIVES: In this study, we quantitatively examined cell density, collagen types I and III, and regional variations in collagen fiber thickness and orientation in the human midmembranous vocal fold lamina propria (LP). METHODS: Lamina propria samples were solubilized with proteinase K or with cyanogen bromide. Cell density was assessed in proteinase K digests by measuring DNA and normalizing it to tissue total protein. Collagen types I and III were quantified by enzyme-linked immunosorbent assay-based detection of collagen type-specific peptides generated by cyanogen bromide digestion. In addition, LP total collagen was determined by measuring sample hydroxyproline levels. Variations in collagen fiber thickness and orientation with LP region were evaluated by examining picrosirius red-stained LP sections with circularly polarized light. RESULTS: The mean (+/-SEM) cell density in the LP and associated epithelium was approximately 0.57 +/- 0.09 million cells per milligram of tissue total protein. Collagen type III composed an average of 34% to 40% of LP total collagen. Quantitative histology indicated that the superficial LP contained an average of 70% thin, 26% intermediate, and 4% thick collagen fibers. This is in contrast to the intermediate and deep LPs, each of which contained less than 25% thin and more than 50% thick collagen fibers. The angular deviations in collagen fiber orientation were relatively large and were similar in magnitude across all LP layers. CONCLUSIONS: The total cell density of the LP and associated epithelium was intermediate between that of hyaline cartilage and dermis. The ratio of collagen type III to total collagen in the LP was similar to that of highly elastic lung parenchyma and roughly twice that of the comparatively less-elastic dermis. The average thickness of collagen fibers increased markedly with increasing LP depth, and the relatively large angular deviations in fiber orientation appeared to correspond in part to the crimped nature of LP collagen fibers.

Constructs for the expression of repeating triple-helical protein domains.

The development of novel scaffolds will be an important aspect in future success of tissue engineering. Scaffolds will preferably contain information that directs the cellular content of constructs so that the new tissue that is formed is closely aligned in structure, composition and function to the target natural tissue. One way of approaching this will be the development of novel protein-based constructs that contain one or more repeats of functional elements derived from various proteins. In the present case, we describe a strategy to make synthetic, recombinant triple-helical constructs that contain repeat segments of biologically relevant domains. Copies of a DNA fragment prepared by PCR from human type III collagen have been inserted in a co-linear contiguous fashion into the yeast expression vector YEpFlag-1, using sequential addition between selected restriction sites. Constructs containing 1, 2 and 3 repeats were designed to maintain the (Gly-X-Y) repeat, which is essential for the formation of an extended triple helix. All constructs gave expressed protein, with the best being the 3-repeat construct which was readily secreted. This material had the expected composition and N-terminal sequence. Incubation of the product at low temperature led to triple-helix formation, shown by reaction with a conformation dependent monoclonal antibody.

Histologic changes of the iris in the development of angle closure in Chinese eyes.

PURPOSE: To identify the histologic characteristics of the iris in primary angle closure. METHODS: Iris specimens were obtained by surgical iridectomy in a series of patients in Zhongshan Ophthalmic Center with newly diagnosed acute angle closure (AAC, 12 eyes), fellow eyes of AAC subjects (FAAC, 14 eyes), primary angle closure suspect (PACS, 11 eyes), chronic angle closure glaucoma (CACG, 19 eyes), primary open angle glaucoma (POAG, 7 eyes), and 9 age-matched donated eyes as controls without evidence of glaucoma. Patients with secondary causes for angle closure were excluded. The sirius red polarization method was used to quantify the density of collagen and differentiate the type I and type III collagen in the iris stroma. Other structural changes were studied by light and electron microscopy. RESULTS: The density of type I collagen was higher in AAC and FAAC eyes (Wilcoxon test, P<0.05), lower in all CACG eyes (Wilcoxon test, P<0.05), and statistically similar in POAG and PACS eyes when compared with normal eyes. Structural damage to the iris stroma was confirmed by light and electron microscopy in AAC, CACG, and POAG eyes but not in the majority of FAAC and PACS eyes. CONCLUSIONS: Eyes that have suffered an acute symptomatic episode of angle closure have a higher density of collagen type I fibers, as do the contralateral fellow eyes. Unlike AAC and CACG eyes, contralateral fellow eyes did not have demonstrable structural damage to the iris. These findings suggest that biomechanical properties of the iris may play a role in the development of AAC attacks.