Ultrastructural localization of Helix pomatia lectin-binding sites in mouse lung elastic fibers

Helix pomatia (Snail) lectin complexed with colloidal gold (HPL-gold) recognized binding sites on elastic fibers in plastic embedded sections of lung tissue from mice of several ages. Deposition of the lectin-gold particles was examined by electron microscopy. Structures such as the elastic laminae of pulmonary vessels and elastic fibers throughout the lung was specifically and intensely decorated by the HPL-gold complex and easily visualized. The binding of the HPL-gold particles was primarily to sites on the amorphous component of elastin, to the virtual exclusion of the microfibrillar elastin elements, collagen fibers and other components of the extracellular matrix. In addition, moderate age differences in the binding of HPL-gold to elastin were apparent. These observations appear to be the first demonstration of the presence, in the amorphous component of elastin, of glycoconjugates that are specifically recognized by HPL and suggest a method by which the involvement of glycoconjugates in lung elastogenesis could be explored.     

Extracellular matrix-mediated remodeling and mechanotransduction in large vessels during development and disease

The vascular extracellular matrix (ECM) is synthesized and secreted during embryogenesis and facilitates the growth and remodeling of large vessels. Proper interactions between the ECM and vascular cells are pivotal for building the vasculature required for postnatal dynamic circulation. The ECM serves as a structural component by maintaining the integrity of the vessel wall while also regulating intercellular signaling, which involves cytokines and growth factors. The major ECM component in large vessels is elastic fibers, which include elastin and microfibrils. Elastin is predominantly synthesized by vascular smooth muscle cells (SMCs) and uses microfibrils as a scaffold to lay down and assemble cross-linked elastin. The absence of elastin causes developmental defects that result in the subendothelial proliferation of SMCs and inward remodeling of the vessel wall. Notably, elastic fiber formation is attenuated in the ductus arteriosus and umbilical arteries. These two vessels function during embryogenesis and close after birth via cellular proliferation, migration, and matrix accumulation. In dynamic postnatal mechano-environments, the elastic fibers in large vessels also serve an essential role in proper signal transduction as a component of elastin-contractile units. Disrupted mechanotransduction in SMCs leads to pathological conditions such as aortic aneurysms that exhibit outward remodeling. This review discusses the importance of the ECM—mainly the elastic fiber matrix—in large vessels during developmental remodeling and under pathological conditions. By dissecting the role of the ECM in large vessels, we aim to provide insights into the role of ECM-mediated signal transduction that can provide a basis for seeking new targets for intervention in vascular diseases.     

Ultrastructural localization of Helix pomatia lectin-binding sites in mouse lung elastic fibers

Summary Helix pomatia (Snail) lectin complexed with colloidal gold (HPL-gold) recognized binding sites on elastic fibers in plastic embedded sections of lung tissue from mice of several ages. Deposition of the lectin-gold particles was examined by electron microscopy. Structures such as the elastic laminae of pulmonary vessels and elastic fibers throughout the lung was specifically and intensely decorated by the HPL-gold complex and easily visualized. The binding of the HPL-gold particles was primarily to sites on the amorphous component of elastin, to the virtual exclusion of the microfibrillar elastin elements, collagen fibers and other components of the extracellular matrix. In addition, moderate age differences in the binding of HPL-gold to elastin were apparent. These observations appear to be the first demonstration of the presence, in the amorphous component of elastin, of glycoconjugates that are specifically recognized by HPL and suggest a method by which the involvement of glycoconjugates in lung elastogenesis could be explored.Supported in part by USPHS Grant HL-32870     

Time course involvement of matrix metalloproteinases in the vascular alterations of renovascular hypertension

Increased vascular matrix metalloproteinases (MMPs) levels play a role in late phases of hypertensive vascular remodeling. However, no previous study has examined the time course of MMPs in the various phases of two-kidney, one-clip hypertension (2K1C). We examined structural vascular changes, collagen and elastin content, vascular oxidative stress, and MMPs levels/activities during the development of 2K1C hypertension. Plasma angiotensin converting enzyme (ACE) activity was measured to assess renin-angiotensin system activation. Sham or 2K1C hypertensive rats were studied after 2, 4, 6, and 10weeks of hypertension. Systolic blood pressure (SBP) was monitored weekly. Morphometry of structural changes in the aortic wall was studied in hematoxylin/eosin, orcein and picrosirius red sections. Aortic NADPH activity and superoxide production was evaluated. Aortic gelatinolytic activity was determined by in situ zymography, and MMP-2, MMP-14, and tissue inhibitor of MMPs (TIMP)-2 levels were determined by gelatin zymography, immunofluorescence and immunohistochemistry. 2K1C hypertension was associated with increased ACE activity, which decreased to normal after 10 weeks. We found increased aortic collagen and elastin content in the early phase of hypertension, which were associated with vascular hypertrophy, increased vascular MMP-2 and MMP-14 (but not TIMP-2) levels, and increased gelatinolytic activity, possibly as a result of increased vascular NADPH oxidase activity and oxidative stress. These results indicate that vascular remodeling of renovascular hypertension is an early process associated with early increases in MMPs activities, enhanced matrix deposition and oxidative stress. Using antioxidants or MMPs inhibitors in the early phase of hypertension may prevent the vascular alterations of hypertension.     

Effect of elastin degradation on carotid wall mechanics as assessed by a constituent-based biomechanical model

Arteries display a nonlinear anisotropic behavior dictated by the elastic properties and structural arrangement of its main constituents, elastin, collagen, and vascular smooth muscle. Elastin provides for structural integrity and for the compliance of the vessel at low pressure, whereas collagen gives the tensile resistance required at high pressures. Based on the model of Zulliger et al. (Zulliger MA, Rachev A, Stergiopulos N. Am J Physiol Heart Circ Physiol 287: H1335-H1343, 2004), which considers the contributions of elastin, collagen, and vascular smooth muscle cells (VSM) in an explicit form, we assessed the effects of enzymatic degradation of elastin on biomechanical properties of rabbit carotids. Pressure-diameter curves were obtained for controls and after elastin degradation, from which elastic and structural properties were derived. Data were fitted into the model of Zulliger et al. to assess elastic constants of elastin and collagen as well as the characteristics of the collagen engagement profile. The arterial segments were also prepared for histology to visualize and quantify elastin and collagen. Elastase treatment leads to a diameter enlargement, suggesting the existence of significant compressive prestresses within the wall. The elastic modulus was more ductile in treated arteries at low circumferential stretches and significantly greater at elevated circumferential stretches. Abrupt collagen fiber recruitment in elastase-treated arteries leads to a much stiffer vessel at high extensions. This change in collagen engagement properties results from structural alterations provoked by the degradation of elastin, suggesting a clear interaction between elastin and collagen, often neglected in previous constituent-based models of the arterial wall.     

Elasticity of passive blood vessels: a new concept

The mechanical properties of passive blood vessels are generally thought to depend on the parallel arrangement of elastin and collagen with linear elasticity and collagen recruitment depending on vessel strain [hook-on (HO) model]. We evaluated an alternative model [serial element (SE) model] consisting of the series arrangement of an infinite number of elements, each containing elastin with a constant elastic modulus and collagen that switches stepwise from slack (zero stress) to fully rigid (infinite stiffness) on ongoing element strain. Both models were implemented with Weibull distributions for collagen recruitment strain (HO model) and collagen tightening strain (SE model). The models were tested in experiments on rat mesenteric small arteries. Strain-tension relations were obtained before and after two rounds of digestion by collagenase. Both models fitted the data prior to digestion. However, for the HO model, this required unrealistically low estimates for collagen recruitment or elastic modulus and unrealistically high estimates for distension of collagen fibers. Furthermore, the data after digestion were far better predicted by the SE model compared with the HO model. Finally, the SE model required one parameter less (collagen elastic modulus). Therefore, the SE model provides a valuable starting point for the understanding of vascular mechanics and remodeling of vessels.     

Elastic laminae in vascular development and disease

The activities of vascular cells, including adhesion, proliferation, and migration, are mediated by extracellular matrix components, including collagen matrix and elastic fibers or laminae. Whereas the collagen matrix stimulates vascular cell adhesion, proliferation, and migration, the elastic laminae inhibit these activities. Coordinated regulation of cell activities by these matrix components is an essential process for controlling the development and remodeling of the vascular system. This article summarizes recent development on the role of arterial elastic laminae in regulating the development of smooth muscle-like cells from bone marrow-derived progenitor cells as well as in mediating cell adhesion, proliferation, and migration with a focus on the molecular mechanisms and physiological significance.     

A Modified Verhoeff-Van Gieson Elastin Histochemical Stain to Enable Pulmonary Arterial Hypertension Model Characterization

Optimal histochemical staining is critical to ensure excellent quality stained sections to enable light microscopic and histomorphometric image analysis. Verhoeff-van Gieson is the most widely used histochemical stain for the visualization of vascular elastic fibers. However, it is notoriously difficult to differentiate fine elastic fibers of small vasculature to enable histomorphometric image analysis, especially in organs such as the lung. A tissue fixation procedure of 10% neutral buffered formalin with subsequent fixation in 70% ethanol further compounds the problem of small vessel staining and identification. Therefore, a modified Verhoeff’s elastin stain was developed as a reliable method to optimally highlight the internal and external elastic laminae of small arteries (50-100 µm external diameter) and intra-acinar vessels (10-50 µm external diameter) in 3 µm thick lung tissue sections from models of pulmonary arterial hypertension. This modified Verhoeff’s elastin stain demonstrated well-defined staining of fine elastic fibers of pulmonary blood vessels enabling subsequent histomorphometric image analysis of vessel wall thickness in small arteries and intra-acinar vessels. In conclusion, modification of the standard Verhoeff-van Gieson histochemical stain is needed to visualize small caliber vessels’ elastic fibers especially in tissues fixed in 10% neutral buffered formalin followed by additional fixation in 70% ethanol.Key words: Histochemical stain, histomorphology, lung, Verhoeff-van Gieson, elastin     

Magnolol inhibits migration of vascular smooth muscle cells via cytoskeletal remodeling pathway to attenuate neointima formation

BackgroundIncreased proliferation and migration of vascular smooth muscle cells (VSMCs) contribute importantly to the formation of both atherosclerotic and restenotic lesions. The objective of this study was to investigate the effect of magnolol on VSMC migration.MethodsThe proteolytic activity of matrix metalloproteinases (MMPs) in tumor necrosis factor alpha (TNF-α) stimulated VSMCs was performed by gelatin zymography. VSMC migration was assessed by wound healing and Boyden chamber methods. Collagen induced VSMC adhesion was determined by spectrofluorimeter and stress fibers formation was evaluated by fluorescence microscope. The expression of signaling molecules involved in stress fibers formation was determined by western blot. The phosphorylation of myosin light chain (MLC20) was determined by urea-glycerol polyacrylamide gel electrophoresis. Immunohistochemistry was performed to determine the expression of β1-integrin and collagen type I in the injured carotid arteries of rats on day 35 after vascular injury.ResultsVSMC migration was strongly inhibited by magnolol without affecting MMPs expression. Also, magnolol inhibited β1-integrin expression, FAK phosphorylation and RhoA and Cdc42 activation to inhibit the collagen induced stress fibers formation. Moreover, magnolol inhibited the phosphorylation of MLC20. Our in vivo results showed that magnolol inhibited β1-integrin expression, collagen type I deposition and FAK phosphorylation in injured carotid arteries without affecting MMP-2 activity.ConclusionsMagnolol inhibited VSMC migration via inhibition of cytoskeletal remodeling pathway to attenuate neointima formation.General significanceThis study provides a rationale for further evaluation of magnolol for the management of atherosclerosis and restenosis.     

Micrometer scale ex vivo multiphoton imaging of unstained arterial wall structure.

BACKGROUND: We characterize the application of multiphoton microscopy to the observation of the extracellular matrix of fresh unstained vessels. METHOD: Combined two-photon-excited fluorescence (2PEF) and second harmonic generation (SHG) imaging of large arteries reveals the architecture of elastin and collagen fibers in the vessel wall with remarkable specificity. RESULTS: We present elastin/collagen imaging in unstained rat vessels at both micrometer and whole vessel scales, and we characterize the optical properties of rat carotid artery and aorta walls. We apply this method to evidence deleterious effects of residual doses of a pesticide on the vessel wall. CONCLUSION: This study illustrates the potential of 2PEF/SHG microscopy for pharmacological studies in unlabeled arteries.     

Role of proteolysis and apoptosis in regression of pulmonary vascular remodeling

Remodeled pulmonary arteries return to normal structural conditions after the increase in pulmonary artery flow resistance is reversed. We studied whether proteolysis of extracellular matrix proteins and apoptosis occur during reversal of remodeling produced by chronic hypoxia in the rat. Main pulmonary arteries were removed at different times during a 10-day period of exposure to 10% O2 and 14 days after return to air. Content and rates of degradation of collagen and elastin as well as immunoreactive collagenase in tissue and isolated mast cells were measured. Immunoblots for collagenase and tissue inhibitor of metalloproteinases (TIMP) were performed. Apoptosis was assessed by cleavage of DNA and TUNEL assay. Excess collagen and elastin present at 10 days of hypoxia decreased to near normal levels after 3-5 days of air. Transient increases in collagenolytic and elastolytic enzyme activities accompanied the rapid decrease in matrix proteins. Mast cells containing collagenase accumulated in remodeled pulmonary arteries, and the active form of collagenase appeared at the time of peak proteolytic activity. TIMP increased during remodeling. Apoptosis was maximal 3 days after return to air. Our results suggest that activation of enzymes, which degrade matrix proteins, and apoptosis play a role in resolution of vascular remodeling.     

Plasmin-dependent and -independent effects of plasminogen activators and inhibitor-1 on ex vivo angiogenesis

Plasminogen activator (PA) inhibitor-1 (PAI-1) has been recognized as a surrogate marker of endothelial dysfunction in diseases associated with impaired angiogenesis, including atherosclerosis, diabetic vasculopathy, and nephropathy. To establish the necessary and sufficient components of the PA system [PAI-1, urokinase-type PA (uPA), or tissue-type PA (tPA), and plasminogen (Plg)] for angiogenesis, we examined angiogenic competence of vascular explant cultures obtained from mice deficient in PAI-1, tPA, uPA, and Plg. To gain insight into the requirement for different matrix-degrading systems during endothelial cell migration across plasmin-degradable basement membranes compared with profibrotic areas containing plasmin-nondegradable collagen, we contrasted vascular sprouting in collagen with Matrigel lattices. PAI-1(-/-) vessels showed an increased capillary sprouting in both collagen and Matrigel. Deficiency of uPA significantly reduced the rate of sprouting, whereas tPA(-/-) vessels showed a profound inhibition of capillary sprouting. The Plg(-/-) vessels failed to sprout, a defect that was restored not only by exogenous Plg, but also by the addition of PAs; a nonproteolytic effect of tPA was observed in Matrigel. Zymography revealed no differences in the activity of metalloproteinase (MMP)-2 and -9 in wild-type and PAI-1(-/-) vessels, but demonstrated reduced MMP-9 activity in all angiogenesis-deficient vessels. In summary, 1) PAI-1 by itself is a modest inhibitor of endothelial sprouting, 2) tPA and Plg are indispensable for angiogenesis in this model, 3) Plg is not the only substrate for PAs, and 4) the activity of MMP-9 is undetectable in explant cultures from tPA and Plg knockout mice.     

Manipulation of remodeling pathways to enhance the mechanical properties of a tissue engineered blood vessel

There is a current need for a small diameter vascular graft due to the limited supply of autogenous grafts and the failure of synthetic grafts due to thrombosis and/or intimal hyperplasia. The use of living cells and tissues to fabricate a small diameter graft (i.e., tissue engineered blood vessel, TEBV) could be useful given the endothelialization potential and biocompatibility benefits of such a graft. However, while sufficient strength has been attained in a TEBV, coordinate compliance has yet to be fine-tuned. In this study we investigate the effects of biological response modifiers, retinoic acid (RA) and ascorbic acid (AA) on TEBV biomechanics as a function of time and subsequently correlate observed RA/AA induced changes in TEBV mechanics with alterations in smooth muscle cell (SMC) biochemistry. TEBVs were constructed using a fibrillar type I collagen network populated by human aortic smooth muscle cells (AoSMC). Following construction this TEBV was treated with 0.3 mM AA and 0.1 mM RA (concentrations found to induce changes in VSMC phenotype). Ultimate tensile stress (UTS), rate of relaxation (RR) and elastic efficiency (EE) of RA/AA treated and untreated TEBVs were measured following 1, 7, 15, 30, 45, and 60 days of treatment. At corresponding time points, the effect of these treatments on collagen and elastin protein synthesis and mRNA expression was examined. RA/AA treated TEBV strength increased and stiffness decreased compared to controls as a function of time. Relative collagen synthesis in treated TEBVs exceeded control levels by nearly two-fold at 15 and 30 days of incubation. RA/AA treated collagen gene expression followed a similar trend. Relative elastin synthesis was also greater in treated TEBVs as compared to untreated TEBVs at 15 and 30 days of incubation and correspondingly elastin mRNA expression was significantly elevated at 15 days of incubation. These data provide evidence that RA/AA treated TEBVs exhibit mechanical properties which more closely mimic those of a native vessel than their untreated counterparts and that changes in extracellular matrix composition and matrix gene expression in the presence of RA/AA treatment may play an important role in the development of said mechanical properties.     

Matrix metalloproteinases and angiogenesis

Matrix metalloproteinases (MMPs) are a family of enzymes that proteolytically degrade various components of the extracellular matrix (ECM). Angiogenesis is the process of forming new blood vessels from existing ones and requires degradation of the vascular basement membrane and remodeling of the ECM in order to allow endothelial cells to migrate and invade into the surrounding tissue. MMPs participate in this remodeling of basement membranes and ECM. However, it has become clear that MMPs contribute more to angiogenesis than just degrading ECM components. Specific MMPs have been shown to enhance angiogenesis by helping to detach pericytes from vessels undergoing angiogenesis, by releasing ECM-bound angiogenic growth factors, by exposing cryptic proangiogenic integrin binding sites in the ECM, by generating promigratory ECM component fragments, and by cleaving endothelial cell-cell adhesions. MMPs can also contribute negatively to angiogenesis through the generation of endogenous angiogenesis inhibitors by proteolytic cleavage of certain collagen chains and plasminogen and by modulating cell receptor signaling by cleaving off their ligand-binding domains. A number of inhibitors of MMPs that show antiangiogenic activity are already in early stages of clinical trials, primarily to treat cancer and cancer-associated angiogenesis. However, because of the multiple effects of MMPs on angiogenesis, careful testing of these MMP inhibitors is necessary to show that these compounds do not actually enhance angiogenesis.     

Processing of VEGF-A by matrix metalloproteinases regulates bioavailability and vascular patterning in tumors

Vascular endothelial growth factor (VEGF) is a critical mediator of blood vessel formation during development and in pathological conditions. In this study, we demonstrate that VEGF bioavailability is regulated extracellularly by matrix metalloproteinases (MMPs) through intramolecular processing. Specifically, we show that a subset of MMPs can cleave matrix-bound isoforms of VEGF, releasing soluble fragments. We have mapped the region of MMP processing, have generated recombinant forms that mimic MMP-cleaved and MMP-resistant VEGF, and have explored their biological impact in tumors. Although all forms induced similar VEGF receptor 2 phosphorylation levels, the angiogenic outcomes were distinct. MMP-cleaved VEGF promoted the capillary dilation of existent vessels but mediated a marginal neovascular response within the tumor. In contrast, MMP-resistant VEGF supported extensive growth of thin vessels with multiple and frequent branch points. Our findings support the view that matrix-bound VEGF and nontethered VEGF provide different signaling outcomes. These findings reveal a novel aspect in the regulation of extracellular VEGF that holds significance for vascular patterning.     

Stability of elastin in the developing mouse aorta: a quantitative radioautographic study

Elastic lamina growth during development and the ultimate stability of elastin in the mouse aortic media was investigated by light and electron microscopic radioautography. Following a single subcutaneous injection of l-[3,4-³H]valine at 3 days of age, animals were killed at 9 subsequent time intervals up to 4 months of age. One day after injection, radioautographic silver grains were primarily observed over the elastic laminae; however, silver grains were also seen over the smooth muscle cells and extracellular matrix. By 21 to 28 days of age, the silver grains were almost exclusively located over the elastic laminae. From 28 days to 4 months of age, the distribution of silver grains appeared relatively unchanged. Quantitation of silver grain number/m² of elastin showed a steady decrease in the concentration of silver grains associated with the elastic laminae from 4 to 21 days of age. After this time, no significant difference in silver grain concentration was observed. Since the initial decrease in grains/m² of elastin corresponds to a period of rapid post-natal growth, the decrease is likely to be a result of dilution of the radiolabel due to new elastin synthesis. With the assumption that little or no significant turnover occurs during this time, a constant growth rate of 4.3% per day was predicted by linear regression analysis. Since no significant difference in the concentration of silver grains was observed from 28 to 118 days of age, no new growth or turnover of elastin can be said to occur during this time period. This is supported by the observation that animals injected with radiolabeled valine at 28 days and 8 months of age showed no significant incorporation of radiolabel into the elastic laminae. The results from this study present the first long-term radioautographic evidence of the stability of aortic elastin and emphasize that initial deposition of elastin and proper assembly of elastic laminae is a critical event in vessel development.     

Paracrine regulation of angiogenesis by different cell types in the aorta ring model

The development of blood vessels during angiogenesis is the result of paracrine interactions between tube-forming endothelial cells and angiogenic factor-producing nonendothelial cells. This process can be reproduced and studied under chemically defined culture conditions by culturing vascular explants in three-dimensional gels of extracellular matrix. Rings of rat or mouse aorta cultured in collagen, fibrin or basement membrane gels produce angiogenic outgrowths composed of a mixed population of endothelial cells and nonendothelial cells. Aortic angiogenesis is regulated by endogenous angiogenic factors, inflammatory cytokines, chemokines, extracellular matrix molecules, and proteolytic enzymes produced by cells of the vessel wall in response to the injury of the dissection procedure. In this paper, we review how macrophages, mural cells and fibroblasts regulate different stages of the angiogenic process, from the formation of immature endothelial sprouts to the reabsorption of the neovessels. We also describe how aortic cultures can be used to study interactions between angiogenic outgrowths and nonvascular cell types such as bone marrow macrophages, platelets or cancer cells. Morphologic, genetic and functional studies of this model have provided invaluable information on how vessels form, mature, interact with nonvascular cell types, and are eventually reabsorbed. Further analysis of the paracrine cross-talk between aortic endothelial and nonendothelial cells is likely to provide new insights into the angiogenic process and its key mechanisms.