Altered shear stress on endothelial cells leads to remodeling of extracellular matrix and induction of angiogenesis.

Endothelial cells (ECs) are subjected to physical forces such as shear stress (SS) induced by blood flow that leads to significant changes in morphology, physiology and gene expression. The abnormal mechanical forces applied in the cardiovascular system can influence the development of conditions and diseases such as thrombosis, hypertension and atherosclerosis. This study investigated the expression of glycosaminoglycans (GAGs), proteoglycans and extracellular matrix molecules in ECs exposed to normal and altered SS. ECs were exposed to SS of 12 dyn/cm2 (artery physiological condition) and 4 dyn/cm2 (artery pathological condition). Subsequently, ECs were subjected to immunofluorescence, qPCR, GAG biosynthesis analyses and cell-based assays. SS induced changes in ECs morphology. There were other pathological consequences of altered SS, including inhibited adhesion, stimulation of migration and capillary-like tube formation, as well as increases of GAG synthesis. We observed higher expression of syndecan-4, perlecan, decorin, fibronectin and collagen III α1 and growth factors, including VEGF-A and TGFß-1. ECs exposed to SS displayed extracellular matrix remodeling as well as expression of cell-matrix and cell-cell interaction molecules. This study contributes to the understanding of how vascular biology is affected by mechanical forces and how these molecules can be affected in cardiovascular diseases.

Mechanical stretch implications for vascular endothelial cells: Altered extracellular matrix synthesis and remodeling in pathological conditions.

AIMS: Cardiovascular diseases such as hypertension, thrombosis and atherosclerosis are responses to mechanical forces applied to the endothelium. Endothelial cells respond to hemodynamic mechanical forces such as cellular mechanical stretching. We investigated the expression of glycosaminoglycans, proteoglycans and other extracellular matrix molecules in endothelial cells subjected to various mechanical stimuli. MAIN METHODS: Endothelial cells were subjected to mechanical stretch in a vacuum system FlexCell™ to 5% (physiological condition) and 15% (pathological condition), for 4 h or 24 h. Culture plates not subjected to strain were used as controls. Subsequently, ECs were subjected to immunofluorescence, real-time PCR, PCR array, glycosaminoglycans biosynthesis using metabolic radiolabeling with 35S-sulfate and cell behavior assays (adhesion, migration and capillary tube formation). KEY FINDINGS: Mechanical stretch induced changes in endothelial cell morphology. Pathological consequences of mechanical stretch included inhibited migration in 2-fold and capillary-like tube formation in 2-fold, when compared to physiological condition after 4 h of ECs exposure; it also reduced total sulfated glycosaminoglycans synthesis thereabout 1.5-fold. Pathological mechanical stretch conditions induced higher expression after 24 h of ECs exposure to mechanical stretch of syndecan-4 (3.5-fold), perlecan (9.1-fold), decorin (5.7-fold), adhesive proteins as fibronectin (5.6-fold) and collagen III α1 (2.2-fold) and growth factors, including VEGF-A (7.3-fold) and TGFß-1 (14.6-fold) and TGFß-3 (4.3-fold). SIGNIFICANCE: Exposure of endothelial cells to mechanical stretch influenced remodeling of the extracellular matrix as well as cell-matrix interactions. These studies improve understanding of how vascular biology is affected by mechanical forces and how these molecules behave in cardiovascular diseases.

Coupling of vinculin to F-actin demands Syndecan-4 proteoglycan.

Syndecans are heparan sulfate proteoglycans characterized as transmembrane receptors that act cooperatively with the cell surface and extracellular matrix proteins. Syn4 knockdown was performed in order to address its role in endothelial cells (EC) behavior. Normal EC and shRNA-Syn4-EC cells were studied comparatively using complementary confocal, super-resolution and non-linear microscopic techniques. Confocal and super-resolution microscopy revealed that Syn4 knockdown alters the level and arrangement of essential proteins for focal adhesion, evidenced by the decoupling of vinculin from F-actin filaments. Furthermore, Syn4 knockdown alters the actin network leading to filopodial protrusions connected by VE-cadherin-rich junction. shRNA-Syn4-EC showed reduced adhesion and increased migration. Also, Syn4 silencing alters cell cycle as well as cell proliferation. Moreover, the ability of EC to form tube-like structures in matrigel is reduced when Syn4 is silenced. Together, the results suggest a mechanism in which Syndecan-4 acts as a central mediator that bridges fibronectin, integrin and intracellular components (actin and vinculin) and once silenced, the cytoskeleton protein network is disrupted. Ultimately, the results highlight Syn4 relevance for balanced cell behavior.

Gradual strenuous running regimen predisposes to osteoarthritis due to cartilage cell death and altered levels of glycosaminoglycans.

OBJECTIVE: To investigate the hypothesis that strenuous running is a predisposing factor for osteoarthritis. DESIGN: Wistar rats were divided into two groups: a control group (CG) and a trained group (TG). The TG underwent a strenuous treadmill running training regimen of controlled intensity, exhibiting progressively improvement of fitness over 12 weeks, running at least 55 km during this period and finally performing an ultra-endurance running exercise to exhaustion. After this period, rats from both groups were euthanized and their knees removed. The articular cartilage was dissected and submitted to histomorphometrical, histomorphological, and immunohistochemical analyses evaluating cell death pathway (caspase-3 and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL)) and inflammatory cytokines [interleukin-1α (IL-1α) and tumor necrosis factor-α (TNF-α)]. In addition, the tissues were analyzed regarding the types and the content of glycosaminoglycans. RESULTS: The TG knee joints exhibited increase in the number of chondrocytes and chondrocyte clusters, as well as significantly increased levels of caspase-3, a protein involved in apoptosis, and of inflammatory cytokines IL-1α and TNF-α. In addition, histologically higher grades of osteoarthritis (Osteoarthritis Research Society International - OARSI grading), and significantly decreased levels of chondroitin sulfate and hyaluronic acid. Knee cartilage thickness and TUNEL did not significantly differ between the two groups. CONCLUSIONS: The articular cartilage of rats subjected to a strenuous running regimen of controlled intensity exhibited molecular and histological characteristics that are present in osteoarthritis.

Quantitative evaluation of experimental choroidal neovascularization by confocal scanning laser ophthalmoscopy: fluorescein angiogram parallels heparan sulfate proteoglycan expression

The objective of the present study was to develop a quantitative method to evaluate laser-induced choroidal neovascularization (CNV) in a rat model using Heidelberg Retina Angiograph 2 (HRA2) imaging. The expression of two heparan sulfate proteoglycans (HSPG) related to inflammation and angiogenesis was also investigated. CNV lesions were induced with argon laser in 21 heterozygous Zucker rats and after three weeks a fluorescein angiogram and autofluorescence exams were performed using HRA2. The area and greatest linear dimension were measured by two observers not aware of the protocol. Bland-Altman plots showed agreement between the observers, suggesting that the technique was reproducible. After fluorescein angiogram, HSPG (perlecan and syndecan-4) were analyzed by real-time RT-PCR and immunohistochemistry. There was a significant increase in the expression of perlecan and syndecan-4 (P < 0.0001) in retinas bearing CNV lesions compared to control retinas. The expression of these two HSPG increased with increasing CNV area. Immunohistochemistry demonstrated that the rat retina damaged with laser shots presented increased expression of perlecan and syndecan-4. Moreover, we observed that the overexpression occurred in the outer layer of the retina, which is related to choroidal damage. It was possible to develop a standardized quantitative method to evaluate CNV in a rat model using HRA2. In addition, we presented data indicating that the expression of HSPG parallels the area of CNV lesion. The understanding of these events offers opportunities for studies of new therapeutic interventions targeting these HSPG.

Quantitative evaluation of experimental choroidal neovascularization by confocal scanning laser ophthalmoscopy: fluorescein angiogram parallels heparan sulfate proteoglycan expression.

The objective of the present study was to develop a quantitative method to evaluate laser-induced choroidal neovascularization (CNV) in a rat model using Heidelberg Retina Angiograph 2 (HRA2) imaging. The expression of two heparan sulfate proteoglycans (HSPG) related to inflammation and angiogenesis was also investigated. CNV lesions were induced with argon laser in 21 heterozygous Zucker rats and after three weeks a fluorescein angiogram and autofluorescence exams were performed using HRA2. The area and greatest linear dimension were measured by two observers not aware of the protocol. Bland-Altman plots showed agreement between the observers, suggesting that the technique was reproducible. After fluorescein angiogram, HSPG (perlecan and syndecan-4) were analyzed by real-time RT-PCR and immunohistochemistry. There was a significant increase in the expression of perlecan and syndecan-4 (P < 0.0001) in retinas bearing CNV lesions compared to control retinas. The expression of these two HSPG increased with increasing CNV area. Immunohistochemistry demonstrated that the rat retina damaged with laser shots presented increased expression of perlecan and syndecan-4. Moreover, we observed that the overexpression occurred in the outer layer of the retina, which is related to choroidal damage. It was possible to develop a standardized quantitative method to evaluate CNV in a rat model using HRA2. In addition, we presented data indicating that the expression of HSPG parallels the area of CNV lesion. The understanding of these events offers opportunities for studies of new therapeutic interventions targeting these HSPG.

A heparin mimetic isolated from a marine shrimp suppresses neovascularization.

BACKGROUND: Choroidal neovascularization (CNV) is the main cause of severe visual loss in age-related macular degeneration (AMD). Heparin/heparan sulfate are known to play important roles in neovascularization due to their abilities to bind and modulate angiogenic growth factors and cytokines. Previously, we have isolated from marine shrimp a heparin-like compound with striking anti-inflammatory action and negligible anticoagulant and hemorrhagic activities. OBJECTIVES: To investigate the role of this novel heparin-like compound in angiogenic processes. METHODS AND RESULTS: The anti-angiogenic effect of this heparinoid in laser-induced CNV and in vitro models is reported. The compound binds to growth factors (FGF-2, EGF and VEGF), blocks endothelial cell proliferation and shows no cytotoxic effect. The decrease in proliferation is not related to cell death either by apoptosis or secondary necrosis. The results also showed that the heparinoid modified the 2-D network organization in capillary-like structures of endothelial cells in Matrigel and reduced the CNV area. The effect on CNV area correlates with decreases in the levels of VEGF and TGF-ß1 in the choroidal tissue. The low content of 2-O-sulfate groups in this heparinoid may explain its potent anti-angiogenic effect. CONCLUSIONS: The properties of the shrimp heparinoid, such as potent anti-angiogenic and anti-inflammatory activities but insignificant anticoagulant or hemorrhagic actions, point to this compound as a compelling drug candidate for treating neovascular AMD and other angioproliferative diseases. A mechanism for the anti-angiogenic effect of the heparinoid is proposed.

Morphological and biochemical evidence of blood vessel damage and fibrinogenolysis triggered by brown spider venom.

The venom of the brown spider is remarkable because it causes dermonecrotic injury, hemorrhagic problems, hemolysis, platelet aggregation and renal failure. The mechanism by which the venom causes hemorrhagic disorders is poorly understood. Rabbits intradermally exposed to the venom showed a local hemorrhage starting 1 h after inoculation and reaching maximum activity between 2 and 3 days. Biopsies examined by light and transmission electron microscopy showed subendothelial blebs, vacuoles and endothelial cell membrane degeneration in blood vessels, plasma exudation into connective tissue, and fibrin and thrombus formation within blood vessels. Loxosceles intermedia venom incubated with fibrinogen partially degrades Aalpha and Bbeta chains of intact fibrinogen, and significantly cleaves all Aalpha, Bbeta and gamma chains when they were separated or when fibrinogen is denatured by boiling. Proteolytic kinetic studies showed that the Aalpha chain is more susceptible to venom hydrolysis than the Bbeta chain. The fibrinogenolysis is blocked by ethylenediamine tetraacetic acid and 1,10-phenanthroline, but not by other protease inhibitors. Human plasma incubated with the venom had coagulation parameters such as prothrombin time, activated partial thromboplastin time and thrombin time increased. Through molecular sieve chromatography, we isolated a venom toxin of 30 kDa with fibrinogenolytic activity. We propose that the local and systemic hemorrhagic disorders evoked in loxoscelism are consequences of direct venom fibrinogenolysis together with cytotoxicity to subendothelial structures and endothelial cells in blood vessels.

Intracolonic bioavailability of human calcitonin in man.

Human calcitonin (hCT) injected into the lumen of the descending colon of normal human subjects was absorbed within minutes and could be recognized intact in plasma as shown by RIA in combination with reverse-phase HPLC. The absorption was low and variable, with bioavailabilities ranging from 0.01% to 2.7% relative to intravenously administered hCT (area under the concentration-time curve). With intravenous hCT serum calcium was lowered and the fractional urinary excretion of calcium, phosphorus, sodium and chloride was significantly stimulated. With the intracolonic hCT, the fractional urinary excretions of calcium, sodium and chloride were also marginally stimulated relative to intracolonic vehicle (placebo). In conclusion, hCT is absorbed intact from the colon, but the bioavailability is low and highly variable.