Total Elastin Research Articles

Copper nanoparticle cues for biomimetic cellular assembly of crosslinked elastin fibers

Elastin, a structural protein distributed in the extracellular matrix of vascular tissues, is critical to maintaining the elastic stability and mechanical properties of blood vessels, as well as regulating cell-signaling pathways involved in vascular injury response and morphogenesis. Pathological degradation of vascular elastin or its malformation within native vessels and the poor ability to tissue-engineer elastin-rich vascular replacements due to innately poor elastin synthesis by adult vascular cells can compromise vascular homeostasis, and must thus be addressed. Our recent studies attest to the utility of hyaluronan (HA) oligomers for elastin synthesis and organization by adult vascular smooth muscle cells (SMCs), though the elastin matrix yields in these cases were quite low relative to total elastin produced. Thus, in this study, we investigated the utility of copper (Cu2+) ions to enhance cellular elastin deposition, crosslinking and maturation into structural fibers. Copper nanoparticles (CuNPs; 80–100nm) in the dose range of 1–100ngml−1 were tested for Cu2+ ion release, and based on mathematical modeling of their release profiles, CuNPs (1, 10, and 400ng ml−1) were chosen for supplementation to adult SMC cultures. The 400ngml−1 dose of CuNPs cumulatively delivered Cu2+ doses in the range of 0.1M, over the 21day culture period. It was observed that while exogenous CuNP supplements do not up-regulate tropoelastin production by vascular SMCs, they promoted formation of crosslinked elastin matrices. The deposition of crosslinked matrix elastin was further improved by the additional presence of HA oligomers in these cultures. Immunofluorescence imaging and structural analysis of the isolated elastin matrices indicate that amorphous elastin clumps were formed within non-additive control cultures, while aggregating elastin fibrils were observed within SMC cultures treated with CuNPs (1–10ngml−1) alone or together with HA oligomers. The presence of 400ngml−1 of CuNPs concurrent with HA oligomers furthered aggregation of these elastin fibrils into mature fibers with diameters ranging from 200 to 500nm. Ultrastructural analysis of elastin matrix within cultures treated with HA oligomers and 400ngml−1 of CuNPs suggest that elastin matrix deposition as stimulated by Cu2+ ions proceeds via a fibrillin-mediated assembly process, with enhanced crosslinking occurring via stimulation of lysyl oxidase. Overall, the data suggest that CuNPs and HA oligomers are highly useful for regenerating crosslinked, fibrillar elastin matrices by adult vascular SMCs. These results have immense utility in tissue-engineering vascular replacements.

The effect of proteolytic treatment on plastic deformation of porcine aortic tissue

The objective of this work was to assess whether selective proteolysis of elastin and/or collagen in a porcine aorta followed by mechanical creep loading would result in an aneurysm-like permanent tissue stretch. The underlying motivations were to (1) test the feasibility of developing an in vitro abdominal aortic aneurysm (AAA) model, and (2) understand what role, if any, that passive creep-induced stretching plays in aneurysmal dilation. Multiple circumferentially oriented flat specimen strips were cut from the porcine thoracic aorta of ten adult pigs. Specimens were subjected to one of six treatment protocols: Untreated controls ( U C ; N = 23 ) , complete elastin degradation ( E ; N = 10 ) , partial elastin degradation ( E p ; N = 10 ) , partial collagen degradation ( C p ; N = 22 ) , and partial degradation of both elastin and collagen ( E p + C p ; N = 3 ) . All specimens were then subjected to cyclic creep (10 min/cycle) with increasing load amplitude until failure. The zero-load strain prior to the creep cycle where failure occurred was defined as load-induced plastic strain. The plastic strain induced by treatment alone, creep loading alone and the total was determined for all specimens. The total plastic strain was significantly greater for E (mean ± SD = 48.2 ±17.6, p<0.005), E p ( 41.6 ± 11.1 , p < 0.0005 ) , but not for E p + C p ( 48.9 ± 21.6 , p = 0.17 ) and C p ( 22.2 ± 12.8 , p = 0.14 ) compared to UC (17.7±6.1). Of the total plastic strain, treatment-induced plastic strain was high for those specimens subjected to partial or total elastin degradation ( E , E p , E p + C p ) , but not for those where elastin was intact ( C p ) . However, load-induced plastic strain in the treated specimens was not different in any of the treated groups compared to controls. Maximum total plastic strain of 78.6% was induced in one porcine aortic tissue from the E group. Even this is far lower than what would be needed for creating a realistic in vitro AAA model. Our findings do not support the feasibility of developing an in vitro AAA by enzymolysis followed by passive stretching of the aorta. The findings also suggest that AAA formation is unlikely to be a passive creep-induced stretching of a proteolytically degraded aortic wall as conventional thinking may suggest, but rather may be predominantly due to growth and remodeling of the aortic wall.

Elastin- and Collagen-Rich Human Carotid Plaques Have Increased Levels of the Cysteine Protease Inhibitor Cystatin C

Background: Cystatin C is a major inhibitor of the elastin- and collagen-degrading cysteine proteases and may therefore have an important role in preserving atherosclerotic plaque stability. In this study we analyzed the associations between human carotid plaque cystatin C expression and the plaque content of collagen and elastin. Methods: Thirty-one plaques were removed by endarterectomy and homogenized. Cystatin C levels were analyzed by densitometry of Western blots and elastin and collagen levels were determined colorimetrically. Results: The plaque content of cystatin C correlated with total elastin (r = 0.58, p = 0.001) and collagen (r = 0.50, p = 0.004), as well as with cross-linked forms of elastin (r = 0.42, p = 0.022) and collagen (r = 0.52, p = 0.003). Immunohistochemical analysis demonstrated that cystatin C colocalized with elastin and collagen. No correlation was seen between cystatin C and the amount of degraded elastin or collagen in plaques. Conclusion: The positive correlation between cystatin C levels and collagen and elastin levels in plaques supports the notion that cystatin C plays an important role in maintaining atherosclerotic plaque stability.

On the in-series and in-parallel contribution of elastin assessed by a structure-based biomechanical model of the arterial wall

Earlier experimental work on decellularized arteries revealed the existence of significant residual stresses within the arterial wall, which are released upon chemical removal of vascular smooth muscle in normal arteries causing substantial radial expansion. Hence, the often-used Hill's model describing active and passive stresses within the wall does not hold true, because the existence of prestresses precludes the fundamental assumption of zero active stress when the vascular smooth muscle is inactive. We have, therefore, developed a new mathematical model based on a modified Hill's model, where the total wall elastin is partitioned in two parts: one in-parallel to vascular smooth muscle and collagen and one connected in-series with vascular smooth muscle. Based on experimental evidences, compressive prestresses were assumed to exist on the parallel elastic component and tensile prestresses on the series elastic component. Further, we assumed that the elastic constants of elastin and collagen and the statistical description of collagen engagement are not affected by decellularization. Excellent fits of the pressure-diameter curves of normal and decellularized arteries were obtained. The model predicts that the majority of elastin is in-series with the vascular smooth muscle (74±8%) and thus only about one-fourth of elastin acts in parallel to the vascular smooth muscle. We conclude that correct biomechanical modeling of the arterial wall requires the knowledge of the zero stress state of both the series and parallel elastic components.

The three-dimensional micro- and nanostructure of the aortic medial lamellar unit measured using 3D confocal and electron microscopy imaging

Changes in arterial wall composition and function underlie all forms of vascular disease. The fundamental structural and functional unit of the aortic wall is the medial lamellar unit (MLU). While the basic composition and organization of the MLU is known, three-dimensional (3D) microstructural details are tenuous, due (in part) to lack of three-dimensional data at micro- and nano-scales. We applied novel electron and confocal microscopy techniques to obtain 3D volumetric information of aortic medial microstructure at micro- and nano-scales with all constituents present. For the rat abdominal aorta, we show that medial elastin has three primary forms: with approximately 71% of total elastin as thick, continuous lamellar sheets, 27% as thin, protruding interlamellar elastin fibers (IEFs), and 2% as thick radial struts. Elastin pores are not simply holes in lamellar sheets, but are indented and gusseted openings in lamellae. Smooth muscle cells (SMCs) weave throughout the interlamellar elastin framework, with cytoplasmic extensions abutting IEFs, resulting in approximately 20° radial tilt (relative to the lumen surface) of elliptical SMC nuclei. Collagen fibers are organized as large, parallel bundles tightly enveloping SMC nuclei. Quantification of the orientation of collagen bundles, SMC nuclei, and IEFs reveal that all three primary medial constituents have predominantly circumferential orientation, correlating with reported circumferentially dominant values of physiological stress, collagen fiber recruitment, and tissue stiffness. This high resolution three-dimensional view of the aortic media reveals MLU microstructure details that suggest a highly complex and integrated mural organization that correlates with aortic mechanical properties.

Elastin Content Correlates With Human Disc Degeneration in the Anulus Fibrosus and Nucleus Pulposus

Quantitative study of elastin content in nondegenerate and degenerate human intervertebral discs. To measure the site-specific changes in elastin content that accompany disc degeneration using a quantitative, dye-binding assay to assess elastin levels. Recently, an abundant and organized network of elastic fibers was observed in nondegenerated human disc using immunostaining histochemistry, suggesting a functional role for elastin. While degenerative changes in the disc extracellular matrix composition are well known, changes in elastin content that may accompany degeneration have not been reported. Human discs were assigned a degenerative grade by 3 independent orthopedic surgeons based on gross morphology. Samples were taken from the outer anulus fibrosus (OAF), inner AF (IAF) and nucleus pulposus (NP). Elastin content was measured using a specific, dye-binding assay and normalized to dry weight and collagen content, which was measured via a hydroxyproline assay. Samples were divided into 2 groups: nondegenerate (Grades 1-2.5) and degenerate (Grades 2.6-4.0). A 2-way analysis of variance was used to test for statistical significance where the 2 factors were disc location and degeneration. Correlations of composition with degeneration and age were analyzed. In nondegenerate tissue, elastin by dry weight was on average 2.0% +/- 0.3%, and there were no differences in elastin content among the locations of OAF, IAF, or NP. With degeneration, there was a significant increase in total disc elastin per dry weight at each location. The degenerate IAF had the largest amount of elastin (9.3% +/- 2.3%), significantly greater than the NP and OAF. Elastin content correlated with degenerative grade and age at each site. Based on the location-dependent degenerative changes, with highest increases in the IAF, elastin may function to restore lamellar structure under radial loads that potentially cause delamination. Future work will focus on distinguishing the changes in elastin orientation with degeneration and understanding the mechanical functional role of elastin in the disc.

Angiotensin Inhibition Decreases Progression of Advanced Atherosclerosis and Stabilizes Established Atherosclerotic Plaques

Although increased extracellular matrix (ECM) is pathogenic in a variety of chronic tissue injuries, reduced and/or disrupted ECM may be detrimental in atherosclerosis and rather destabilize existing atherosclerotic lesions. This study therefore assessed the effects of angiotensin II (AngII) antagonism on ECM components of advanced atherosclerosis. Twenty-four-week-old apolipoprotein E-deficient mice were treated with the AngII antagonist losartan for 12 wk. Controls received water or hydralazine. AngII antagonism significantly reduced progression of established atherosclerosis, whereas hydralazine showed no benefit despite similar decrease in BP. Although there was no difference in the macrophage component, AngII antagonism increased the relative collagen portion of the lesions; lessened elastin fragmentation, increased the total elastin content of the aorta; and reduced the mRNA and activity/protein of the elastolytic proteases, cathepsin S, and metalloproteinase-9. Extracellular elastin degradation by cultured smooth muscle cells (SMC) was reduced by losartan, as was SMC invasion through an elastin gel barrier. Thus, AngII antagonism lessens progression of atherosclerosis, increases collagen, and preserves elastin components of ECM within the vascular lesions, which, at least in part, is modulated by effects on SMC. These effects not only decrease further expansion of advanced lesions but also stabilize the established atherosclerotic plaques and may underlie the decreased incidence of acute cardiovascular events that are observed in patients in whom AngII antagonism is begun after atherosclerosis is already established.

The role of neutrophil elastase in elastin metabolism of pelvic tissues from women with stress urinary incontinence

Altered elastin metabolism is implicated in pelvic floor disorders. We studied neutrophil elastase (NE) and matrix metalloproteinase (MMP) activities in vaginal tissues from premenopausal women with stress urinary incontinence (SUI). Elastase and NE activities in vaginal tissues were assessed. Protein and mRNA expressions were determined by RT-PCR and Western blot. Total elastin and collagen contents were evaluated. To compare the relative elastolytic effect of NE and MMP-2, we used their respective antibodies to immunoprecipitate these proteins from vaginal fibroblast extracts prior to assessing elastase activity. Elastase activity in vaginal wall tissues was significantly higher in the secretory compared to the proliferative phase. NE mRNA and protein expressions were similar between control and SUI tissues from the secretory phase. However, NE activity in the SUI tissues was higher compared to control tissues. The mRNA expression of alpha-1 antitrypsin (ATT) was higher in control tissues from the proliferative phase compared to those from the secretory phase, while no difference was observed in SUI tissues between either phase. Protein expression of the active form of ATT was decreased in SUI tissues compared to controls during the secretory phase. Anti-NE antibody reduced total elastase activity by 60-70%, compared to less than 20% reduction with anti-MMP-2 antibody. During the secretory phase, elastolytic activity is increased in pelvic tissues from women with SUI, through an increase in NE activity and a concurrent decrease in ATT expression. The serine protease, NE, appears to be a more significant modulator of elastase activity compared to MMP-2.

Maternal undernutrition reduces aortic wall thickness and elastin content in offspring rats without altering endothelial function

Epidemiological studies suggest a link between fetal/early infant nutrition and adult coronary artery disease. In the present study, we examined the effects of altering nutrition during gestation, lactation and juvenile life on aortic structure and function in rats. Wistar rat dams were fed either a control or low-protein diet throughout pregnancy, or a low-protein diet for the final 7 days of gestation only. At 21 days post-partum, male pups were weaned on to a control, low-protein or high-fat diet. At 12 weeks, the offspring rats were killed. In 46 rats, aortic sections were mounted and stained to assess media thickness and elastin content. In a further 38 rats, aortic rings were suspended in an organ bath and vascular reactivity was tested with dose-response curves to the endothelium-dependent dilator acetylcholine and the endothelium-independent dilator sodium nitroprusside. Rats exposed to maternal protein restriction while in utero had a significantly decreased aortic wall thickness compared with control rats (P=0.005). Total elastin content of the aorta was also decreased by both maternal low-protein (P=0.02) and early postnatal low-protein (P=0.01) diets. Neither maternal nor postnatal low-protein or high-fat diets, however, resulted in any significant changes in arterial dilator function. In conclusion, fetal undernutrition in rats, induced via a maternal low-protein diet, causes a decrease in aortic wall thickness and elastin content without altering aortic dilator function. These changes in vascular structure may amplify aging-related changes to the vasculature and contribute to the pathophysiology of the putative link between impaired fetal growth and adult cardiovascular disease.

In Vivo Attenuation of Myointimal Hyperplasia Using Transforming Growth Factor-Beta3 in an Interposition Graft Model

To examine if transforming growth factor-beta3 (TGFbeta3) can attenuate the development of para-anastomotic myointimal hyperplasia in an animal model of small-diameter vascular graft failure. Under general anesthesia, 10 adult goats underwent bilateral polyurethane interposition graft insertion in the carotid position. Following completion of the anastomoses, each artery received adventitial infiltration of 50 ng of TGFbeta3 around the anastomoses; a placebo was administered to the other side. Postoperatively, each animal received 150 mg of aspirin daily. The arteries were explanted, half at 6 weeks and the remaining 5 at 3 months, for histological examination. Vessel wall thickness surrounding the anastomosis was reduced by 37% in TGFbeta3-treated arteries compared to placebo at 6 weeks and 3 months, principally due to reduced smooth muscle cell proliferation. There was decreased overall luminal loss on angiography. Total collagen content was not significantly different between TGFbeta3 and placebo sides. Further analysis for the subendothelial matrix component collagen type VIII showed decreased levels on the treated side. Total elastin content was reduced on the TGFbeta3-treated side. Direct single-dose subadventitial infiltration of TGFbeta3 following insertion of an interposition graft reduces SMC proliferation and elastin content. It would appear that TGFbeta3 holds promise as a prophylaxis against the development of myointimal hyperplasia, the predominant cause of graft failure in peripheral bypass surgery.

Select bladder smooth muscle cell functions were enhanced on three-dimensional, nano-structured poly(ether urethane) scaffolds

Bladder wall resection is often required as a treatment for invasive bladder cancer. When this happens, a suitable replacement material is needed. The present study, therefore, created three-dimensional, porous, nano-structured poly(ether urethane) (PU) matrices for use as bladder tissue-engineering scaffolds. Select cytocompatibility experiments (specifically adhesion and long-term growth studies) were performed on these scaffolds using human bladder smooth muscle cells (BdSMCs). In addition, the amount of total collagen and elastin present in each cell-seeded scaffold was determined since the production of these extracellular matrix (ECM) proteins is essential for the health and survival of cells and for the functionality of the replaced organ. Finally, to better understand how these scaffolds and resident cells would perform in the complex mechanical environment of the bladder wall, scaffolds and cells were subjected to 10 cmH2O pressure using a computer-controlled pressure chamber. Results provided evidence that compared to conventionally used, micro-dimensional PU scaffolds, the novel, nanodimensional scaffolds created in this research increased cell adhesion, growth, and ECM protein production. Additionally, scaffolds and resident cells were not affected by exposure to 10 cmH2O pressure (compared to controls maintained under atmospheric conditions). These results are promising and provide evidence that the nano-dimensional PU scaffolds created in this research are suitable bladder replacement materials that may outperform materials currently used for such purposes.

Reduction of myointimal hyperplasia after arterial anastomosis by local injection of transforming growth factor β3

The transforming growth factor (TGF)-beta family of cytokines exerts pleiotropic actions on vascular smooth muscle cell phenotype, proliferation, and extracellular matrix synthesis. This in vivo study assessed the use of TGF-beta3 in attenuating the development of postanastomotic smooth muscle cell proliferation. Under general anesthesia, 10 adult goats underwent transection and reanastomosis of both common carotid arteries. After reanastomosis, one artery was infiltrated with 50 ng of TGF-beta3 in 100 microL of pH buffer around the anastomosis, and the other side was infiltrated with buffer only. After surgery, each animal received 150 mg of aspirin daily. The arteries were explanted after 3 months for histologic examination. Vessel wall thickness surrounding the anastomosis was reduced by 30% after TGF-beta3 treatment compared with placebo (P = .003), with a 20% (P = .002) reduction in cellular content. Although total collagen content was not significantly different between TGF-beta3 and placebo, collagen type VIII content was reduced around the TGF-beta3 anastomoses (P = .011). A reduction in the total elastin content (P = .003) and number of elastic fiber lamellae (P = .042) was found surrounding TGF-beta3-treated anastomoses, but not placebo-treated anastomosis. A 29% increase in vasa vasorum (P = .044) was present around TGF-beta3-treated anastomoses. No differences in inflammatory cell infiltration were seen between sides. Direct subadventitial infiltration of TGF-beta3 immediately after creation of an arterial anastomosis attenuates cell proliferation, with a reduction in elastin and collagen type VIII content and vessel wall thickness.

Skin biopsy analysis reveals predisposition to stretch mark formation

Stretch marks are a disfiguring skin condition of yet unknown etiology. We wanted to compare the functional disposition of dermal fibroblasts, derived from unaffected skin of patients with stretch marks and fibroblasts from normal age-matched subjects, and develop a test to predict predisposition to stretch mark development. Skin biopsies from normal subjects (NS), stretch-marked skin (SM), and normal-looking skin from patients with stretch marks (NL) were analyzed by histochemistry and assays of total protein, DNA, and elastin. Cellular migration, proliferation, and matrix production were also measured in primary cultures of biopsy-derived fibroblasts. We found that NL skin contained less DNA, protein, and elastin than NS skin (-16%, -36%, -44%, respectively) and that such deficiencies were more profound in SM skin (-55%, -64%, -80%, respectively). Both NL- and SM-derived cells had slower than normal outgrowth of their fibroblasts, which also demonstrated low migration and proliferation rates, and produced less elastin, fibrillin 1, collagen 1, and fibronectin than NS-derived cells in primary cultures. All these aberrant features, indicating a dormant phenotype of NL- and SM-derived fibroblasts, were reversed and normalized in the fourth passage of all tested fibroblasts. A series of in vitro tests led to the discovery of a dormant phenotype in dermal fibroblasts from patients with stretch marks. The described tests may serve as a diagnostic tool for predicting predisposition to stretch marks. The reported reversibility of impaired fibroblast phenotypes opens a new perspective for preventive treatments for people predisposed to stretch mark formation.

Short-term treatment of spontaneously hypertensive rats with liver growth factor reduces carotid artery fibrosis, improves vascular function, and lowers blood pressure

Liver growth factor (LGF), a mitogen for liver cells, reduces fibrosis in a rat model of cirrhosis. The present study assesses the possible vascular antifibrotic and antihypertensive effects of LGF treatment on spontaneously hypertensive rats (SHR). Six-month-old male SHR and normotensive Wistar Kyoto rats (WKY) were treated with LGF (4.5 microg LGF/rat i.p. twice a week for 2 weeks). Haemodynamic parameters were measured in anaesthetized rats. Vascular structure and function were studied in carotid arteries using optical and confocal microscopy, radioimmunoassay for desmosine, and isometric tension recording. LGF reduced systolic and diastolic blood pressure only in SHR. When compared to those of untreated SHR, carotid arteries from LGF-treated SHR showed: 1) a 50% reduction in collagen area and an increase in vascular smooth muscle cell number in the media, 2) no difference in total elastin content, but an increase in size of fenestrae in the internal elastic lamina, and 3) enhanced relaxation to acetylcholine, sodium nitroprusside, and forskolin. These effects were specific for SHR, since no changes were observed in LGF-treated WKY. Short-term treatment with a low dose of LGF induced a large improvement in vascular structure and function and significantly reduced blood pressure in a rat model of essential hypertension. The present results could open future research to explore the vascular effects of this endogenous factor in order to determine its potential as an antifibrotic and antihypertensive agent in humans.

Elastin and Calcium Rather Than Collagen or Lipid Content Are Associated With Echogenicity of Human Carotid Plaques

Echolucent carotid plaques have been associated with increased risk for stroke. Histological studies suggested that echolucent plaques are hemorrhage- and lipid-rich, whereas echogenic plaques are characterized by fibrosis and calcification. This is the first study to relate echogenicity to plaque composition analyzed biochemically. Echogenicity of human carotid plaques was analyzed by standardized high-definition ultrasound and classified into echolucent, with gray-scale median (GSM) <32 and echogenic with GSM > or =32. The biochemical composition of the plaques was assessed by fast-performance liquid chromotography and high-performance thin-layer chromotography. As assessed biochemically (milligrams per gram [mg/g]), echolucent plaques contained less hydroxyapatite (43.8 [SD 41.2] mg/g versus 121.6 [SD 106.2] mg/g; P=0.018), more total elastin (1.7 [SD 0.4] mg/g versus 1.2 [SD 0.4] mg/g; P=0.008), and more intermediate-size elastin forms (1.2 [SD 0.3] mg/g versus 0.8 [SD 0.4] mg/g; P=0.018). There was no difference in collagen amount between echogenic and echolucent plaques, neither biochemically (15.3 [SD 3.7] mg/g versus 14.4 [SD 3.4] mg/g) nor histologically (13.4 [SD 4.9] % versus 13.0 [SD 5.6] %). Cholesterol esters, unesterified cholesterol, and triglycerides were increased in plaques associated with symptoms (22.5 [SD 23.3] mg/g versus 13.3 [SD 3.2]; P=0.04), but no differences were detected between echolucent and echogenic plaques (13.5 [SD 4.0] versus 20.2 [SD 21.5] mg/g). Similar results were obtained by Oil Red O staining (symptomatic 7.6 [SD 4.7] % versus asymptomatic 4.2 [SD 3.6] %; P=0.03; echolucent 5.9 [SD 4.1] % versus echogenic 5.0 [SD 4.0] % of area). Echogenicity of carotid plaques is mainly determined by their elastin and calcium but not collagen or lipid content. In addition, echolucency is associated to higher elastin content.

Aneurysm Growth, Elastinolysis, and Attempted Doxycycline Inhibition of Elastase-induced Aneurysms in Rabbits

To establish the relationship between elastin degradation and aneurysm growth in New Zealand white rabbit model aneurysms, and to explore the potential for pharmacologic inhibition of elastinolysis and aneurysm growth with use of the matrix metalloproteinase (MMP) inhibitor doxycycline. Elastase-induced, saccular aneurysms created in the right common carotid artery in 30 animals randomly divided into controls (n = 16) and doxycycline treated (n = 14) were studied. Aneurysm growth was determined by angiography and aneurysm specimens were collected at 7 and 14 days for histologic and immunohistochemical analysis. Aneurysms were characterized by marked elastin degradation and thickening of the arterial wall media in the absence of inflammatory cell markers. There was no evidence for expression of MMPs in the aneurysm wall at any time point. Aneurysm formation and growth were not prevented by the systemic administration of doxycycline. Mean aneurysm width increased from 3.1 +/- 0.7 mm at 3 days to 3.7 +/- 0.8 mm at 7 days and 4.2 +/- 0.8 mm at 14 days (P =.012 and P =.017, respectively). There was no statistically significant difference in aneurysm size and elastin content at any time point between doxycycline treated and control animals. Elastase-induced rabbit aneurysm formation is accompanied by total elastin destruction that was not inhibited by the administration of doxycycline. Aneurysms in this model may be caused by the initial infusion of elastase, rather than by ongoing degradation from endogenous proteases released by inflammatory cells.

Sustained changes in lung expansion alter tropoelastin mRNA levels and elastin content in fetal sheep lungs.

Our objective was to determine the effects of sustained alterations in fetal lung expansion on pulmonary elastin synthesis. In fetal sheep, lung expansion was either decreased between 111 and 131 days' gestation (term approximately 147 days) by tracheal drainage or increased for 2, 4, 7, or 10 days by tracheal obstruction, ending at 128 days' gestation. Lung tropoelastin mRNA levels were assessed by Northern blot analysis, total elastin content was measured biochemically, and staining of lung sections was used to assess the localization and form of elastic fibers. Tracheal obstruction significantly elevated pulmonary tropoelastin mRNA levels 2.5-fold at 2 days, but values were not different from controls at 4, 7, and 10 days; elastin content tended to be increased at all time points. A sustained decrease in lung expansion by tracheal drainage reduced pulmonary tropoelastin mRNA levels 2.5-fold; elastin content was also decreased compared with controls, and tissue localization was altered. Our results indicate that the degree of lung expansion in the fetus influences elastin synthesis, content, and tissue deposition.

Aminoguanidine and aortic wall mechanics, structure, and composition in aged rats.

With aging, the aortic wall becomes stiffer. This could be because of changes in wall stress or composition. We investigated whether a specific change in wall composition, ie, accumulation of advanced glycation end products (AGEs) on the extracellular matrix, is a major factor. We measured aortic mechanics, geometry, and composition in 3-, 10-, 15-, 20-, and 30-month-old inbred normotensive Wistar-Glaxo/Rijswick rats and in a group of 30-month-old rats treated from 20 months onward with aminoguanidine (AG, 42 mg/kg per day), an inhibitor of AGE formation. Thoracoabdominal aortic (pressure) pulse-wave velocity (PWV) increased progressively with age (44% from 3 to 30 months). This age-related increase in aortic PWV was not related to changes in wall stress. For all ages, central (and peripheral) aortic mean blood pressures were not statistically different. Dilatation occurred (18% increase in internal diameter from 3 to 30 months), but this was accompanied by outward hypertrophic remodeling, with an increase in the medial cross-sectional area of 95% and in the ratio of medial thickness to internal diameter of 29%. Wall stress decreased with age (-34%). There was an increase in the ratio of elastic modulus (calculated from the Moens-Korteweg equation) to wall stress (calculated from the Lamé equation, 117% from 3 to 30 months), suggesting that a change in the composition of the wall is responsible for the age-linked increase in wall stiffness. Dry weight decreased slightly but significantly (-14%) with age. Total protein, elastin, collagen, and nonscleroprotein protein [total-(elastin+collagen)] contents did not change with age, but calculated densities of all 4 were halved (as the medial cross-sectional area doubled). The elastin/collagen ratio was statistically similar at all ages. The only significant effect of AG treatment was a fall in PWV (-20%), leading to a fall in the elastic modulus/wall stress ratio (-27% at 10 months of AG treatment versus 30 months of no treatment). In conclusion, the age-related increase in aortic wall stiffness is prevented by 10 months of treatment with AG, which has no effect on wall stress or composition, suggesting that AG may improve aortic wall stiffness by lowering the degree of AGE-induced cross-linking of the extracellular matrix scleroproteins, such as collagen.

Gelatinase B Is Required for Alveolar Bronchiolization After Intratracheal Bleomycin Instillation

Increased expression of matrix metalloproteinases, particularly gelatinase B (matrix metalloproteinase-9), has been described in the lungs in pulmonary fibrosis. Intratracheal bleomycin is often used experimentally to produce lesions resembling human fibrosing alveolitis. To assess the role of gelatinase B in bleomycin-induced fibrosing alveolitis, we instilled bleomycin intratracheally into gelatinase B-deficient mice and gelatinase B +/+ littermates. Twenty-one days after bleomycin instillation, the two groups of mice were indistinguishable in terms of pulmonary fibrosis histologically and total lung collagen and elastin. However, the lungs of gelatinase B-deficient mice showed minimal alveolar bronchiolization, while bronchiolization was prominent in the lungs of gelatinase B +/+ mice. Gelatinase B was identified immunohistochemically in terminal bronchiolar cells and bronchiolized cells 7 days and 14 days after bleomycin instillation in gelatinase B +/+ mice, and whole-lung gelatinase B messenger RNA was increased at the same time. Many bronchiolized cells displayed Clara cell features by electron microscopy and by immunostaining with antibody to Clara cell-specific protein. Some bronchiolized cells stained with antibody to forkhead/winged helix transcription factor 4, a factor associated with the ciliated cell phenotype, but none stained for an alveolar type-II cell marker, surfactant protein C. Thus, fibrosing alveolitis develops after intratracheal bleomycin instillation irrespective of gelatinase B. However, gelatinase B is required for alveolar bronchiolization, perhaps by facilitating migration of Clara cells and other bronchiolar cells into regions of alveolar injury.

Gelatinase B Is Required for Alveolar Bronchiolization after Intratracheal Bleomycin

Increased expression of matrix metalloproteinases, particularly gelatinase B (MMP-9), has been described in the lungs in pulmonary fibrosis. Intratracheal bleomycin is often used experimentally to produce lesions resembling human fibrosing alveolitis. To assess the role of gelatinase B in bleomycin-induced fibrosing alveolitis, we instilled bleomycin intratracheally into gelatinase B-deficient mice and gelatinase B+/+ littermates. Twenty-one days after bleomycin the two groups of mice were indistinguishable in terms of pulmonary histology and total lung collagen and elastin. However, the lungs of gelatinase B-deficient mice showed minimal alveolar bronchiolization, whereas bronchiolization was prominent in the lungs of gelatinase B+/+ mice. Gelatinase B was identified immunohistochemically in terminal bronchiolar cells and bronchiolized cells 7 and 14 days after bleomycin in gelatinase B+/+ mice, and whole lung gelatinase B mRNA was increased at the same times. Many bronchiolized cells displayed Clara cell features by electron microscopy. Some bronchiolized cells stained with antibody to helix transcription factor 4, a factor associated with the ciliated cell phenotype. Thus, fibrosing alveolitis develops after intratracheal bleomycin irrespective of gelatinase B. However, gelatinase B is required for alveolar bronchiolization, perhaps by facilitating migration of Clara cells and other bronchiolar cells into the regions of alveolar injury.