Elastin Staining Research Articles

DONOR-REACTIVE ALLOANTIBODY PRODUCTION CORRELATES WITH NEITHER ALLOGRAFT SURVIVAL NOR DEVELOPMENT OF TRANSPLANT VASCULAR SCLEROSIS (TVS)

151 Introduction: Patients can develop donor-reactive antibodies post-transplant, although the pathologic significance of these are controversial. Donor-reactive antibodies often develop in murine cardiac allograft recipients that accept grafts due to short-term treatment with agents such as anti-CD4 mAb or gailium nitrate (GN), but are frequently not investigated. This study characterizes the generation of donor-reactive alloantibodies in mice that have accepted cardiac allografts, and relates these alloantibodies to the incidence of graft acceptance and to the development of transplant vascular sclerosis (TVS). Methods: DBA/2 cardiac allografts were heterotopically implanted into C57BL/6 recipients, which were treated peri-transplant with the anti-CD4 mAb, GK1.5, or with gallium nitrate (GN) for 28 days to permit long-term graft survival. At 60 days post transplant, sera were collected and the allografts harvested from recipients with functioning grafts. Donor-reactive alloantibodies were assessed by flow cytometry. The extent of TVS was determined using trichromic and elastin staining of sections from the midsection of the heart. Results: All of the animals in this study had graft survival>60 days, while untreated recipients rejected their grafts in 7-10 days. Approximately 80% of GN-treated and 50% of GK1.5-treated allograft recipients developed donor-reactive IgG alloantibodies. In animals which developed IgG alloantibodies, titers generally were >1/250, and all IgG isotypes were represented, although alloantibodies of the IgG1 isotype were the most prevalent in GN-treated recipients, with few animals producing high levels of IgG2a or IgG2b. GK1.5-treated recipients did not exhibit any prevalence of isotype production. Interestingly, all of the grafts developed TVS and interstitial fibrosis, regardless of the antibody level or isotype. Conclusions: Our data indicate that avoidance of acute rejection does not imply a lack of allosensitization, since donor-reactive, IgG alloantibodies are present in most long term allograft recipients. Additionally, neither the amount, nor the IgG subclass of donor-reactive alloantibody correlates with graft acceptance or with the development of TVS or fibrosis. Thus, donor-reactive alloantibodies are not indicative of either graft acceptance or rejection, nor are they indicative of TVS development.

Doxycycline inhibits elastin degradation and reduces metalloproteinase activity in a model of aneurysmal disease

Purpose: Abdominal aortic aneurysms are characterized by degradation of the extracellular matrix, with a reduction in the elastin concentration of the arterial media. These changes are mediated by increased levels of endogenous metalloproteinases (MMPs) within the aorta, which provide a potential therapeutic target for pharmacologic agents aimed at reducing the growth rate of small aneurysms. In this study, the ability of doxycycline—an MMP inhibitor—to reduce matrix degradation was assessed in a previously described model of aneurysmal disease that used a brief pulse of elastase to induce MMP production and elastin degradation in arterial organ cultures. Methods: Porcine aortic segments (n = 8) were preincubated in exogenous pancreatic elastase for 24 hours before culture in standard conditions for 13 days with both 1 and 10 mg/L doxycycline. Control segments were cultured both without doxycycline and without elastase. At the termination of culture, MMPs were extracted from the tissue and quantified by a combination of substrate gel enzymography and immunoblotting. The volume fractions of elastin and collagen were determined by stereologic analysis of sections stained with Miller's elastin and van Gieson's stain. Results: Stereologic analysis demonstrated a significant preservation of elastin in aorta treated with doxycycline 10 mg/L ( p < 0.001) and demonstrated that this preservation was accompanied by a significant reduction in MMP-9 activity ( p < 0.02). Immunoblotting for tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) showed no decreased production in the doxycycline-treated groups. Conclusions: Therapeutic ranges of doxycycline significantly inhibited elastin degradation and MMP-9 production within aortic organ cultures. These data suggest that doxycycline may have a potential application in reducing the growth rates of small abdominal aortic aneurysms. (J Vasc Surg 1998;27:354-61.)

Wound healing: A paradigm for lumen narrowing after arterial reconstruction

Purpose: The intimal hyperplasia hypothesis that equates lumen narrowing after arterial injury with intimal mass has recently been challenged. Evidence has emerged to suggest that lumen narrowing is caused in large part by changes in artery wall geometry rather than intimal mass per se. We have begun to explore this hypothesis in a unique nonhuman primate model of atherosclerosis. Methods: Monkeys who were fed an atherogenic diet for 3 to 5 years underwent experimental angioplasty of the left iliac artery. The contralateral iliac artery served as an intraanimal control. Arteries were removed 2, 4, 7, 14, 28, or 112 days later for analysis (6 or 13 per time point). Angioplasty dilated arteries by fracturing atheroma and stretching or tearing the media. Cross-sections of injured arteries were analyzed for expression of extracellular matrix components and cell surface integrins that are important in wound healing. Antibodies, riboprobes, or histochemical stains specific for fibrin, hyaluronan, versican (chondroitin sulfate–containing proteoglycan), procollagen-I, elastin, and the α 2β 1 and α Vβ 3 integrins were used. Results: A thin mural thrombus was seen at sites of denudation and plaque fracture (days 2 to 7). This provisional matrix was invaded by leukocytes (days 2 to 4) and α-actin–positive smooth muscle cells (SMCs; days 4 to 7). Thrombus was replaced by SMCs expressing hyaluronan and the associated versican proteoglycans (day 14). Versican was expressed throughout the neointima as it enlarged (day 28), but expression later subsided (day 112). Procollagen-I expression initially increased in the adventitia (day 4) and then in the forming neointima (day 14). Procollagen-I expression was found to persist within the adventitia and in the neointima in SMCs nearest the lumen (days 28 to 112). Elastin staining was prominent within the mature neointima (day 112) but not at earlier time points. Integrin expression also increased within the injured artery wall. α Vβ 3 staining (fibrin[ogen] receptor) increased in the injured media (days 2 to 7) and was then seen throughout the early neointima (day 7). Low level expression of α Vβ 3 subsequently persisted within the forming neointima (day 28). α 2β 1 (collagen receptor) expression increased in the neointima in SMCs nearest the lumen (day 28). Conclusions: Lumen narrowing after angioplasty in this model of atherosclerosis is caused largely by decreased artery wall diameter. The pattern of matrix and integrin expression within the injured artery wall is in many ways analogous to that of healing wounds. These observations suggest that tissue contraction may play a role in lumen narrowing at sites of arterial reconstruction. Strategies to inhibit wound contraction may prove effective in preventing restenosis. (J Vasc Surg 1998;27:96-108.)

Staining for Elastin and Collagen

Staining for Elastin and Collagen

Smooth muscle cell proliferation, elastin formation, and tropoelastin transcripts during the development of intimal thickening in rabbit carotid arteries after endothelial denudation.

Extracellular matrix formation and smooth muscle cell proliferation are two major factors contributing to the development of intimal thickening after arterial injury. We investigated the elastin formation, tropoelastin transcripts, and proliferation of smooth muscle cells during the development of intimal thickening in rabbit carotid arteries after balloon endothelial denudation. Tropoelastin transcripts, identified by in situ hybridization using a digoxigenin-labeled probe, and elastin staining in the thickened intima were minimal 1 week after endothelial denudation when smooth muscle cell proliferation appeared throughout the thickened intima. A strong signal for tropoelastin transcripts was seen in the basal layer of the thickened intima 2 weeks after endothelial denudation, and then in the surface layer of the thickened intima 4 weeks after endothelial denudation. Immunohistochemistry for proliferating cell nuclear antigen and Ki-67, both markers for proliferating cell nuclei, showed that tropoelastin transcripts and elastin formation increased when smooth muscle cells enter quiescence after the end of the proliferative phase in the intima. Our findings strongly suggest that elastin synthesis and smooth muscle cell proliferation are tightly regulated during the repair of arterial wall injury.

Modification of Movat Pentachrome Stain with Improved Reliability of Elastin Staining

Modification of Movat Pentachrome Stain with Improved Reliability of Elastin Staining

Modification of Movat Pentachrome Stain with Improved Reliability of Elastin Staining

AbstractThe authors describe a modification of Movat pentachrome stain for the demonstration of elastin, sulfated macromolecules (proteoglycans, glycosaminoglycans and secreted mucins), collagens, myofibrils, and erythrocytes. The key modification is the use of a modified Verhoeff elastic tissue stain, which is more tolerant of minor variations in technique and does not require differentiation to a subjective endpoint. The resulting stained sections have a clear background and crisply defined cell and matrix components including both large and small elastic fibers. This stain is particularly useful for evaluating the matrix components of lung and vascular tissue in normal and diseased states. (The J Histotechnol 19:325–327, 1996)

Extracellular matrix characterization during healing of full-thickness wounds treated with a collagen/elastin dermal substitute shows improved skin regeneration in pigs.

We investigated the architecture of the extracellular matrix (ECM) during healing of full-thickness wounds in the pig. Two different treatments, one based on epidermal transplantation (split skin mesh grafts, SP wounds) and one consisting of a combination of epidermal transplantation and a dermal matrix substitute (MA wounds) were compared. The dermal matrix consisted of native bovine collagen coated with elastin hydrolysate. The latter treatment reduced wound contraction and improved tissue regeneration. The expression patterns of fibronectin, von Willebrand factor, laminin, chondroitin sulfate, and elastin, detected by immunohistochemistry, were examined in time and indicated different stages of healing. During the early phase of healing the dermal matrix induced more granulation tissue, a different fibronectin expression pattern, and rapid vascular cell ingrowth (von Willebrand factor). Furthermore, in the MA wounds chondroitin sulfate was detected earlier in the basement membrane and fibronectin staining disappeared more rapidly. During later stages of healing, chondroitin sulfate expression was selective for areas in which ECM remodeling was active; in these specific areas elastin staining reappeared. ECM remodeling and elastin regeneration occurred both in the upper and lower dermis for the MA wounds but only in the upper dermis for the SP wounds. Electron microscopic evaluation of the wounds after 2 weeks showed many myofibroblasts in the SP wounds, whereas in the MA wounds cells associated with the dermal matrix had characteristics of normal fibroblasts. The results suggest that the biodegradable dermal matrix served as a template for dermal tissue regeneration, allowed faster regeneration, and improved the quality of healing in large full-thickness skin defects.

Angiogenesis in abdominal aortic aneurysms

To determine the degree of neovascularisation in the wall of abdominal aortic aneurysms in comparison to atherosclerotic control aortas, and to correlate the angiogenic response with the extent of the cellular inflammatory infiltrate. Histopathological study. Aortic samples were obtained from patients with abdominal aortic aneurysms and from atherosclerotic controls. Samples were stained with haematoxylin and eosin, and Miller's elastin and Van Gieson stain, EVG, and a monoclonal antibody specific to human endothelial cells. Within the aortic wall three histological regions were defined, the media, the adventitia and a transition zone. The number of capillary like, thin walled vessels were measured in each region, and the cellular infiltrate was quantified. The number of newly formed vessels was increased in all layers of aneurysmal wall in comparison to control samples (p<0.001). The degree of neovascularisation correlated with the extent of the inflammatory infiltrate (rs=0.45, p<0.01). This study demonstrated that abdominal aortic aneurysms are associated with a marked angiogenic response, which is related to the degree of inflammation within the aortic wall. It is hypothesised that anti-angiogenic agents may play a role in the medical management of aortic aneurysmal disease.

The association of human fibulin-1 with elastic fibers: an immunohistological, ultrastructural, and RNA study.

We examined the pattern of fibulin-1 mRNA and protein expression in human tissues and cell lines. Fibulin-1 transcripts were found in RNA isolated from most tissues and a variety of cultured cells, including fibroblasts, smooth muscle cells, and several epithelial cell lines, but not endothelial cells, lymphomyloid cells, or a number of carcinoma and melanoma lines. Immunohistochemical analysis showed that fibulin-1 is an intercellular component of connective tissues, predominantly associated with matrix fibers in tissues such as the cervix, dermis, intimal and medial layers of blood vessels, heart valves, meningeal tissue of the brain, Wharton's jelly of the umbilical cord, testis, and lung. Most of the fibers that were immunoreactive with fibulin-1 antibodies also stained with antibodies to the elastic fiber proteins elastin and fibrillin, as well as with Verhoeff's elastin stain. Immunoelectron microscopic analysis of elastin fibers of skin and saphenous vein revealed that fibulin-1 was located within the amorphous core of the fibers, similar to elastin, but it was not in the fibrillin-containing, elastin-associated microfibrils. Our finding that fibulin-1 is an elastic fiber component suggests several possible new functions for fibulin-1, e.g., that it is a structural protein that contributes to the elastic properties of connective tissue fibers or that is involved with the process of fibrogenesis.

Simple testicular cyst in the neonate

The authors report the first case of a simple testicular cyst presenting in a neonate. Diagnostic criteria are outlined and theories concerning etiology are discussed. The authors show an absence of elastin staining in the specimen, and suggest this precludes germinal tubules as a source. The rationale for testicular-sparing surgery is discussed.

Histopathologic Features of the Floppy Eyelid Syndrome Involvement of Tarsal Elastin

Purpose: Patients with the floppy eyelid syndrome have chronic papillary conjunctivitis with easily everted upper eyelids and a soft, pliant upper tarsus. The purpose of this study is to describe the clinical features and the histopathologic correlate in a group of patients with floppy eyelid syndrome. Methods: The authors examined eight patients with floppy eyelid syndrome, four of whom underwent surgical management with horizontal eyelid shortening. Eyelid tissue from these patients was examined using light microscopy, electron microscopy, and immunohistochemistry and compared with controls with unrelated eyelid or orbital disorders. Results: Clinical findings included obesity or eye rubbing, lash ptosis, and, less commonly, blepharoptosis. Two patients had documented sleep apnea with abnormal sleep electroencephalogram. Light microscopy of the surgical specimens showed chronic conjunctival inflammation, papillary conjunctivitis, and meibomian gland abnormalities, including granuloma formation. Verhoeff's modified elastin stain demonstrated a marked decrease in the amount of elastin fibers in tarsus from patients with floppy eyelid syndrome compared with controls. Immunohistochemical staining for elastin also showed a marked decrease of tarsal elastin in floppy eyelid patients compared with controls. In contrast, immunohistochemical stains showed that the distribution of collagen types I and III was similar between patients with floppy eyelid syndrome and controls. Electron microscopy demonstrated that tarsal collagen was comparable in patients and controls, and that there was a reduced amount of tarsal elastin in floppy eyelid syndrome compared with controls. Conclusions: These findings demonstrate that tarsal elastin is decreased in the floppy eyelid syndrome, which may contribute to the laxity of the tarsus in this disorder.

1004-54 Correlation of Intravascular Ultrasound Imaging with Histology of Normal and Diseased Pulmonary Arteries

Intravascular ultrasound (IVUS) imaging of pulmonary arteries may provide useful clinical information to assess the degree of vascular disease. To investigate the feasibility of IVUS to identify the morphological changes associated with pulmonary vascular disease, 21 arterial segments (diameter range 2–6 mm) were obtained at autopsy and formalin fixed. IVUS imaging was performed with a 25 MHz mechanical catheter. Elastin and trichrome stains were used to grade the collagen and elastin content. IVUS and histology (Hist) images were digitized and measured. An intensity index was used to quantitate the IVUS appearance based on the average intensity per pixel and the standard deviation of intensities. Based on the histologic appearance, the arterial segments were categorized into normal (n = 6), mild hyperplasia (n = 9), and moderate hyperplasia (n = 6). Although the lumen size was easily identified, IVUS imaging revealed only a single layer appearance despite the presence of intimal hyperplasia. In distinction to coronary and peripheral arteries, IVUS did not identify a 2 or 3 layer appearance in the pulmonary arteries. This homogeneous IVUS pattern was due to the high elastin or collagen content of the intima, media and adventitia. HIST IVUS r p Lumen Perimeter 9.3 ± 2.8 10.4 ± 3.4 0.96 &lt;0.0001 Lumen Area 7.5 ± 5.0 5 ± 6.7 0.97 &lt;0.0001 NL (n = 6) Mild (n = 9) Mod (n = 6) P (ANOVA) Intensity Index 0.58 ± 0.08 0.65 ± 0.11 0.76 ± 0.05 &lt;0.01 IVUS accurately measures the lumen size of pulmonary arteries. It is unable to identify the degree of intimal hyperplasia because of the homogenous echogenicity across the different layers of the artery. The echo intensity index may be useful in distinguishing normal from diseased pulmonary arteries.

Histopathologic Features of the Floppy Eyelid Syndrome: Involvement of Tarsal Elastin

Patients with the floppy eyelid syndrome have chronic papillary conjunctivitis with easily everted upper eyelids and a soft, pliant upper tarsus. The purpose of this study is to describe the clinical features and the histopathologic correlate in a group of patients with floppy eyelid syndrome. The authors examined eight patients with floppy eyelid syndrome, four of whom underwent surgical management with horizontal eyelid shortening. Eyelid tissue from these patients was examined using light microscopy, electron microscopy, and immunohistochemistry and compared with controls with unrelated eyelid or orbital disorders. Clinical findings included obesity or eye rubbing, lash ptosis, and, less commonly, blepharoptosis. Two patients had documented sleep apnea with abnormal sleep electroencephalogram. Light microscopy of the surgical specimens showed chronic conjunctival inflammation, papillary conjunctivitis, and meibomian gland abnormalities, including granuloma formation. Verhoeff's modified elastin stain demonstrated a marked decrease in the amount of elastin fibers in tarsus from patients with floppy eyelid syndrome compared with controls. Immunohistochemical staining for elastin also showed a marked decrease of tarsal elastin in floppy eyelid patients compared with controls. In contrast, immunohistochemical stains showed that the distribution of collagen types I and III was similar between patients with floppy eyelid syndrome and controls. Electron microscopy demonstrated that tarsal collagen was comparable in patients and controls, and that there was a reduced amount of tarsal elastin in floppy eyelid syndrome compared with controls. These findings demonstrate that tarsal elastin is decreased in the floppy eyelid syndrome, which may contribute to the laxity of the tarsus in this disorder.

The histopathology of infrainguinal vein graft stenoses

The precise histopathological nature of vein graft stenoses is unclear. Some authors have suggested that these lesions are due to intimal hyperplasia and others have claimed that they are fibrous strictures. The aim of this study was to determine the histological nature of infrainguinal vein graft stenoses by examining sections of vein grafts that had developed stenoses and had been surgically revised. This was performed using a combined anti-smooth muscle actin/Millers elastin stain. The results show that vein graft stenoses are due to intimal hyperplasia whereby smooth muscle cells proliferate and cause thickening of the intimal layer.

Effects of Glutaraldehyde on Experimental Arterial Iso- and Allografts in Rats

The effects of glutaraldehyde pretreatment and allograft rejection in arterial grafts were assessed, using iso- and allografts in rats. An in situ glutaraldehyde fixation procedure was used to obtain homogeneous cross-linked vascular biografts. Ten Lewis rats were isografted, ten were isografted with a glutaraldehyde-treated aortic segment, ten were allografted with aortic segments from brown Norway (BN) inbred rats, and ten were allografted with glutaraldehyde-treated BN aortas. The macroscopic and microscopic appearances of the grafts were analyzed 3 weeks after the initial surgery. Immunological injury to the media and the intimal response were quantified morphometrically after monochromatic staining of cell nuclei (hematoxylin after periodic acid), elastin (orcein), and calcification (Von Kossa). Untreated isografts were normal. Untreated allografts showed the classical signs of arterial wall rejection: adventitial inflammatory granuloma, reduced medial thickness and smooth muscle cell density, and greatly increased intimal thickness ( P < 0.005). Glutaraldehyde treatment significantly decreased the medial thickness in both iso- and allografts ( P < 0.001) and prevented the intimal proliferative response ( P < 0.005), but did not change adventitial inflammation. It also induced massive calcification mainly in isografts ( P < 0.001). Histomorphological modifications of glutaraldehyde-treated grafts are consistent with a partial protective effect of glutaraldehyde against the rejection process, but also with an induction of a nonspecific inflammatory reaction. Glutaraldehyde-induced cross-linking of the extracellular matrix was responsible for ectopic calcification of the arterial grafts which was independent of the rejection process.

Extracellular matrix changes of the optic nerve lamina cribrosa in monkey eyes with experimentally chronic glaucoma.

Using light microscopic immunohistochemistry, we studied the immunolocalization and immunoreactivity of the extracellular matrix, including collagen types III, IV, VI, laminin, and alpha elastin in the lamina cribrosa of monkey eyes with normal and experimentally chronic glaucoma. Our results showed: (1) abnormal linearlike immunodeposits of both collagen type IV and laminin in the margin of the lamina cribrosa with significant density in the glaucomatous eyes; (2) the immunoreactivity of collagen type III resembled that of the normal eye, but was slightly stronger at the laminar surface; (3) findings with collagen type VI resembled those of type III with an enhanced linearlike staining surrounding the nerve-fiber bundles. Furthermore, staining of alpha elastin demonstrated dramatic changes in both reactivity and localization. The lamina cribrosa of glaucomatous eyes showed a markedly reduced immunoreactivity as well as an irregular, interrupted pattern. These observations suggest that the changes might be a secondary to the long-standing elevation of intraocular pressure. The alteration of these macromolecules may modify the course of glaucomatous optic damage.

Effect of perindopril on the immune arterial wall remodeling in the rat model of arterial graft rejection

A model of arterial graft arteriosclerosis is described in which arterial wall immune injury was induced by grafting segments of abdominal aorta between two histologically incompatible strains of rats. The effect of hypertension and its treatment with the angiotensin-converting enzyme (ACE) inhibitor perindopril was tested using inbred spontaneously hypertensive rats (SHR) and their normotensive controls (Wistar-Kyoto [WKY]). Each of the grafted hypertensive and normotensive rats was randomly allocated to placebo treatment (10 SHR, 10 WKY) and perindopril treatment (2 mg/kg/day) (10 SHR, 10 WKY). The immune injury and the arterial wall response were quantified morphometrically 2 months after the grafting using specific stains for collagen, elastin, and nuclei. Hypertension was associated with a significant increase in intimal thickness. Treatment with perindopril greatly reduced intimal proliferation, decreasing the intimal thickness and the collagen content within the intimal layer. In contrast, hypertension and ACE inhibition had little effect on the arterial wall injury. We conclude that hypertension and its treatment with perindopril significantly affect graft arteriosclerosis. These effects seem to be independent of their effects on arterial wall injury, but not independent of blood pressure.

Elastin at the Hinge Portion of the Eustachian Tube Cartilage in Specimens from Normal Subjects and Those with Cleft Palate

The density of elastin in the intermediate portion between the lateral lamina and the medial lamina of human eustachian tube (ET) cartilage was examined in six normal adults, seven normal children, and six children with cleft palate (CP) in order to obtain information about how the physical properties of the ET cartilage differ as a function of age and presence of CP. Cross sections of the midcartilaginous portions of the ETs that had been stained with Weigert's elastin stain were photographed at uniform magnification, the area for study was projected, and the meshlike are of the ET cartilage that stained elastin-positive was represented on paper by lines. A digitizer was used to measure the total length of all the lines representing elastin in each photomicrograph, and the mean was determined for each of the three groups. The mean density of elastin was significantly greater in normal adults than in normal children (Student's t test, t = 2.781; p less than .02). It was also significantly greater in normal children than in CP children (Wilcoxon t = 24.0; p less than .05). These results appear to indicate that CP children have poorer elasticity in this area of the ET cartilage, which might cause functional obstruction (floppiness) of the ET in those children.

Quantitative Determination of Murine Dermal Elastic Fibers by Color Image Analysis: Comparison of Three Staining Methods

We compared three different staining methods to determine if the dermal elastic fiber content of the HRS/Skh-1 hairless mouse could be accurately measured by color image analysis. Comparisons were made among Kligman's modification of Luna's mast cell stain for elastin, Unna's orcein stain with or without potassium permanganate preoxidation, and Gomori's aldehyde fuchsin stain with potassium permanganate preoxidation. The color image analysis system could be used to identify and quantify murine dermal elastin fibers in sections stained by all three methods. Gomori's aldehyde fuchsin stain with preoxidation demonstrated twice the content of dermal elastic fibers demonstrated by either Kligman's modification of Luna's mast cell stain or Unna's orcein stain with or without preoxidation. Gomori's aldehyde fuchsin method with preoxidation should be considered the stain of choice for evaluating murine dermal elastic fiber content.