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.

Is HSPG2 a modifier gene for Marfan syndrome?

Marfan syndrome (MFS) is a connective tissue disease caused by variants in the FBN1 gene. Nevertheless, other genes influence the manifestations of the disease, characterized by high clinical variability even within families. We mapped modifier loci for cardiovascular and skeletal manifestations in the mg∆loxPneo mouse model for MFS and the synthenic loci in the human genome. Corroborating our findings, one of those loci was identified also as a modifier locus in MFS patients. Here, we investigate the HSPG2 gene, located in this region, as a candidate modifier gene for MFS. We show a correlation between Fbn1 and Hspg2 expression in spinal column and aorta in non-isogenic mg∆loxPneo mice. Moreover, we show that mice with severe phenotypes present lower expression of Hspg2 than those mildly affected. Thus, we propose that HSPG2 is a strong candidate modifier gene for MFS and its role in modulating disease severity should be investigated in patients.

Is the Expression of the Components of the Carotid Matrix of Rats Influenced by Estrogen, Progestin and Tibolone?

OBJECTIVE: To analyze the effects of estrogen alone or in combination with progestogens and tibolone (TIB) on the expression of the extracellular matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9), of perlecan, and of heparanase (HPSE) of the vascular walls of the carotid arteries. METHODS: A total of 30 250-day-old ovariectomized Wistar rats were orally treated for 5 weeks with: a) 1 mg/kg of estradiol benzoate (EB); b) EB + 0.2 mg/kg of medroxyprogesterone acetate (MPA); c) EB + 0.2mg/kg of norethisterone acetate (NETA); d) EB + 2 mg/kg of dydrogesterone (DI); e) 1 mg/kg of TIB; f) placebo (CTR). Following treatment, the expression of mRNA for MMP-2, MMP-9, and HPSE was analyzed by real-time polymerase chain-reaction (PCR), and the expression of MMP-2, of MMP-9, of tissue inhibitor of metalloproteinase 2 (TIMP-2), and of perlecan was quantified by immunohistochemistry in the carotid arteries. RESULTS: The groups showed significant differences on mRNA HPSE expression (p = 0.048), which was higher in the EB, EB + MPA, and TIB groups. There was no statistically significant difference in mRNA MMP-2 or MMP-9 expression. The immunohistochemical expression of MMP-2, of TIMP-2, of MMP-9, of HPSE, and of perlecan showed no differences between groups. CONCLUSION: Estradiol alone or associated with MPA and TIB treatment can increase mRNA HSPE expression of the walls of the carotid arteries in ovariectomized rats.

OBJETIVO: Analisar os efeitos do estrogênio isolado ou em combinação com progestogênios e tibolona (TIB) na expressão das metaloproteinases 2 e 9 da matriz extracelular (MMP-2 e MMP-9), da perlecan e da heparanase (HPSE) das paredes vasculares das artérias carótidas. MéTODOS: Trinta ratas Wistar ovariectomizadas com 250 dias de idade foram tratadas oralmente por 5 semanas com: a) 1 mg/kg de benzoato de estradiol (EB); b) EB + 0,2 mg/kg de acetato de medroxiprogesterona (MPA); c) EB + 0,2mg/kg de acetato de noretisterona (NETA); d) EB + 2 mg/kg de didrogesterona (DI); e) 1 mg/kg de TIB; f) placebo (CTR). Após o tratamento, a expressão de mRNA para MMP-2, MMP-9, e HPSE foi analisada por reação em cadeia da polimerase (RCP) em tempo real, e a expressão de MMP-2, MMP-9, inibidor tecidual de metaloproteinase 2 (TIMP-2), e de perlecan foi quantificado por imunohistoquímica em artérias carótidas. RESULTADOS: Os grupos apresentaram diferenças significativas na expressão do mRNA HPSE (p = 0,048), sendo maiores nos grupos EB, EB + MPA e TIB. Não houve diferença estatisticamente significativa nas expressões de mRNA MMP-2 ou MMP-9. A expressão imunohistoquímica de MMP-2, TIMP-2, MMP-9, HPSE e perlecan não mostrou diferenças entre os grupos. CONCLUSãO: O estradiol isolado ou associado ao tratamento com MPA e TIB pode aumentar a expressão de mRNA HSPE nas paredes das artérias carótidas em ratas ovariectomizadas.

Is the expression of the components of the carotid matrix of rats influenced by estrogen, progestin and tibolone? / A expressão dos componentes da matriz carotídea de ratos é influenciada pelo estrogênio, progestagênio e tibolona?

Abstract Objective To analyze the effects of estrogen alone or in combination with progestogens and tibolone (TIB) on the expression of the extracellular matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9), of perlecan, and of heparanase (HPSE) of the vascular walls of the carotid arteries. Methods A total of 30 250-day-old ovariectomized Wistar rats were orally treated for 5 weeks with: a) 1 mg/kg of estradiol benzoate (EB); b) EB + 0.2 mg/kg of medroxyprogesterone acetate (MPA); c) EB + 0.2mg/kg of norethisterone acetate (NETA); d) EB + 2 mg/kg of dydrogesterone (DI); e) 1 mg/kg of TIB; f) placebo (CTR). Following treatment, the expression of mRNA for MMP-2, MMP-9, and HPSE was analyzed by realtime polymerase chain-reaction (PCR), and the expression of MMP-2, of MMP-9, of tissue inhibitor of metalloproteinase 2 (TIMP-2), and of perlecan was quantified by immunohistochemistry in the carotid arteries. Results The groups showed significant differences on mRNA HPSE expression (p = 0.048), which was higher in the EB, EB + MPA, and TIB groups. There was no statistically significant difference in mRNA MMP-2 or MMP-9 expression. The immunohistochemical expression of MMP-2, of TIMP-2, of MMP-9, of HPSE, and of perlecan showed no differences between groups. Conclusion Estradiol alone or associated with MPA and TIB treatment can increase mRNA HSPE expression of the walls of the carotid arteries in ovariectomized rats.

Resumo Objetivo Analisar os efeitos do estrogênio isolado ou em combinação com progestogênios e tibolona (TIB) na expressão das metaloproteinases 2 e 9 da matriz extracelular (MMP-2 e MMP-9), da perlecan e da heparanase (HPSE) das paredes vasculares das artérias carótidas. Métodos Trinta ratas Wistar ovariectomizadas com 250 dias de idade foram tratadas oralmente por 5 semanas com: a) 1 mg/kg de benzoato de estradiol (EB); b) EB + 0,2 mg/kg de acetato de medroxiprogesterona (MPA); c) EB + 0,2mg/kg de acetato de noretisterona (NETA); d) EB + 2 mg/kg de didrogesterona (DI); e) 1 mg/kg de TIB; f) placebo (CTR). Após o tratamento, a expressão de mRNA para MMP-2, MMP- 9, e HPSE foi analisada por reação em cadeia da polimerase (RCP) em tempo real, e a expressão de MMP-2, MMP-9, inibidor tecidual de metaloproteinase 2 (TIMP-2), e de perlecan foi quantificado por imunohistoquímica em artérias carótidas. Resultados Os grupos apresentaram diferenças significativas na expressão do mRNA HPSE (p = 0,048), sendo maiores nos grupos EB, EB + MPA e TIB. Não houve diferença estatisticamente significativa nas expressões de mRNA MMP-2 ou MMP-9. A expressão imunohistoquímica de MMP-2, TIMP-2, MMP-9, HPSE e perlecan não mostrou diferenças entre os grupos. Conclusão O estradiol isolado ou associado ao tratamento com MPA e TIB pode aumentar a expressão de mRNA HSPE nas paredes das artérias carótidas em ratas ovariectomizadas.

Heparan sulfate proteoglycans as trastuzumab targets in anoikis-resistant endothelial cells.

Anoikis is a form of programmed cell death induced by loss of contact from neighboring cells or from their extracellular matrix (ECM). Many tumorigenic cells are anoikis resistant, facilitating cancer progression and metastasis. Trastuzumab is a monoclonal antibody used for the treatment of breast and gastric cell cancer, but its mechanism of action is not well elucidated and its target molecules not well defined. Heparan sulfate proteoglycans (HSPGs) and glycosaminoglycans (GAGs) play important roles in tumor development and in response of cancer cells to drugs. This study investigates the effect of trastuzumab on the expression of HSPGs and sulfated glycosaminoglycans (SGAGs) in anoikis-resistant endothelial cells. After trastuzumab treatment, endothelial cells resistant to anoikis show an increase in adhesion to fibronectin followed by a decrease in invasion, proliferation, and angiogenic capacity. In addition, a significant increase in the number of cells in the S phase of the cell cycle was also observed. In relation to HSPGs and SGAGs expression, we observed a decrease in syndecan-4 and perlecan expression, as well as in the heparan sulfate biosynthesis in anoikis-resistant endothelial cells after exposure to trastuzumab. Our results suggest that trastuzumab interacts with GAGs and proteoglycans of the cell surface and ECM and through this interaction controls cellular events in anoikis-resistant endothelial cells.

The role of SDF1 in prostate epithelial morphogenesis.

Androgens induce rat prostate induction from the urogenital sinus epithelium at embryonic day 17.5. Subsequent morphogenesis, including epithelial cord growth, branching, and canalization, results from concerted paracrine interactions with the stroma. A significant number of paracrine factors bind heparan sulfate (HS). We hypothesized that interfering with overall sulfation could disrupt the signaling mediated by HS-binding factors and that the undersulfated environment would allow investigation of individual exogenous morphogens. First, we investigated whether acinar morphogenesis involved HS-proteoglycan expression and found that syndecans 1 and 3 were upregulated in RWPE1 cells in the transition from two- to three-dimensional (3D) Matrigel, capable of promoting spheroid formation. We then investigated whether sodium chlorate, a general sulfation inhibitor, interfered with spheroid formation by RWPE1 cells and acinar morphogenesis in ex vivo ventral prostate (VP) organ culture. As expected, treatment with sodium chlorate inhibited spheroid formation by RWPE1 cells in 3D culture. Chlorate also inhibited ex vivo VP epithelial branching and canalization, resulting in long branchless epithelial structures. We then investigated whether the HS-binding factors, FGF10, TGFß1, and SDF1, could reverse the effect of sodium chlorate. Although no effect was seen in the FGF10- and TGFß1-treated samples, SDF1 promoted epithelial canalization in the low sulfated environment, highlighting its specific role in lumen formation. Altogether, the results show that sodium chlorate perturbed prostate morphogenesis and allowed investigation of factors involved in branching and/or canalization, implicating SDF1 signaling in epithelial canalization.

Heparan Sulfate Proteoglycans in Human Colorectal Cancer.

Colorectal cancer is the third most common cancer worldwide, accounting for more than 610,000 mortalities every year. Prognosis of patients is highly dependent on the disease stage at diagnosis. Therefore, it is crucial to investigate molecules involved in colorectal cancer tumorigenesis, with possible use as tumor markers. Heparan sulfate proteoglycans are complex molecules present in the cell membrane and extracellular matrix, which play vital roles in cell adhesion, migration, proliferation, and signaling pathways. In colorectal cancer, the cell surface proteoglycan syndecan-2 is upregulated and increases cell migration. Moreover, expression of syndecan-1 and syndecan-4, generally antitumor molecules, is reduced. Levels of glypicans and perlecan are also altered in colorectal cancer; however, their role in tumor progression is not fully understood. In addition, studies have reported increased heparan sulfate remodeling enzymes, as the endosulfatases. Therefore, heparan sulfate proteoglycans are candidate molecules to clarify colorectal cancer tumorigenesis, as well as important targets to therapy and diagnosis.

Heparan Sulfate Proteoglycans May Promote or Inhibit Cancer Progression by Interacting with Integrins and Affecting Cell Migration.

The metastatic disease is one of the main consequences of tumor progression, being responsible for most cancer-related deaths worldwide. This review intends to present and discuss data on the relationship between integrins and heparan sulfate proteoglycans in health and cancer progression. Integrins are a family of cell surface transmembrane receptors, responsible for cell-matrix and cell-cell adhesion. Integrins' main functions include cell adhesion, migration, and survival. Heparan sulfate proteoglycans (HSPGs) are cell surface molecules that play important roles as cell receptors, cofactors, and overall direct or indirect contributors to cell organization. Both molecules can act in conjunction to modulate cell behavior and affect malignancy. In this review, we will discuss the different contexts in which various integrins, such as α5, αV, ß1, and ß3, interact with HSPGs species, such as syndecans and perlecans, affecting tissue homeostasis.

Epithelial basement membrane proteins perlecan and nidogen-2 are up-regulated in stromal cells after epithelial injury in human corneas.

The epithelial basement membrane (BM) is a specialized extracellular matrix that has been shown to have a critical role in corneal development, wound healing, and disease. Although the epithelial BM contributes to corneal homeostasis, relatively little is know about non-epithelial production of its components that may be important in defective regeneration of the epithelial basement membrane associated with opacity after photorefractive keratectomy. The purpose of the current study was to investigate stromal production of corneal epithelial BM proteins in wounded human corneas using immunohistochemistry. A total of five unwounded control eyes and five 30-min epithelial-wounded corneas were obtained from fresh corneoscleral buttons removed from human eyes enucleated due to choroidal melanoma with normal anterior segments. In the wounded corneas, an eight mm patch of central corneal epithelium and epithelial BM was removed with a Beaver blade when the patient was under general anesthesia. Immunohistochemical analyses were performed to detect perlecan and nidogen-2 proteins-important components of the epithelial BM lamina lucida and lamina densa zones. Perlecan and nidogen-2 proteins were detected in the BM itself and at low levels in keratocytes in all unwounded corneas. After epithelial injury, both perlecan and nidogen-2 were expressed at high levels in stromal keratocytes, including superficial keratocytes in the early phases of apoptosis. Thus, after epithelial and epithelial BM injury, stromal keratocytes contribute important perlecan and nidogen-2 components to the regenerating epithelial BM.

Mechanisms of cellular uptake, intracellular transportation, and degradation of CIGB-300, a Tat-conjugated peptide, in tumor cell lines.

CIGB-300 is a cyclic synthetic peptide that induces apoptosis in malignant cells, elicits antitumor activity in cancer animal models, and shows tumor reduction signs when assayed in first-in-human phase I trial in patients with cervical tumors. CIGB-300 impairs phosphorylation by casein kinase 2 through targeting the substrate's phosphoacceptor domain. CIGB-300 was linked to the cell penetrating peptide Tat to facilitate the delivery into cells. Previously, we showed that CIGB-300 had a differential antiproliferative behavior in different tumor cell lines. In this work, we studied differential antiproliferative behavior in terms of cellular uptake, intracellular transportation, and degradation in tumor cell lines with dissimilar sensitivity to CIGB-300. The internalization of CIGB-300 was studied in different malignant cell lines. We found that the cell membrane heparan sulfate proteoglycans act as main receptors for extracellular CIGB-300 uptake. The most sensitive tumor cell lines showed higher intracellular incorporation of CIGB-300 in comparison to less sensitive cell lines. Furthermore, CIGB-300 uptake is time- and concentration-dependent in all studied cell lines. It was shown that CIGB-300 has the ability to penetrate cells mainly by direct membrane translocation. However, a minor proportion of the peptide uses an energy-dependent endocytic pathway mechanism to gain access into cells. CIGB-300 is internalized and transported into cells preferentially by caveolae-mediated endocytosis. Lysosomes are involved in CIGB-300 degradation; highly sensitive cell lines showed degradation at earlier times compared to low sensitive cells. Altogether, our data suggests a mechanism of internalization, vesicular transportation, and degradation for CIGB-300 in tumor cells.

Effect of estrogen therapy on vascular perlecan and metalloproteinases 2 and 9 in castrated rats.

AIM: To study the effects of estrogen therapy on the expression of matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) and perlecan in the vascular wall. METHODS: Twenty 180-day-old Wistar rats were castrated and treated 1 week later for a period of 4 weeks with one of the following: (1) placebo; (2) 0.5 µg/day estradiol benzoate (E(2)B); (3) 5 µg/day E(2)B; (4) 50 µg/day E(2)B. A fifth group consisted of rats that had not been castrated. Following treatment, expression of MMP-2 and MMP-9 mRNA (MMP-2([RNA]) and MMP-9([RNA]), respectively) was analyzed by real-time PCR, and expression of MMP-2 (MMP-2([IH])), MMP-9 (MMP-9([IH])) and perlecan was quantified by immunohistochemistry, in carotid walls. RESULTS: There were no differences among castrated groups for MMP-2([RNA]) (p = 0.1969) and for MMP-9([RNA]) (p = 0.1828); however, a correlation was observed between E(2)B dose and MMP-9([RNA]) levels (r = 0.471, p = 0.018). Differences among groups were observed for MMP-2([IH]), MMP-9([IH]) and perlecan (p < 0.0001), wherein higher levels were observed in animals treated with estrogen therapy, correlating with E(2)B doses in the case of MMP-9 (r = 0.441, p = 0.026) and perlecan (r = 0.574, p = 0.005). CONCLUSIONS: Estrogen therapy correlates with higher levels of MMP-2, MMP-9 and perlecan in the extracellular matrix of carotid walls in castrated rats, in a dose-dependent manner. There was a dose-response effect of E(2)B on the expression of MMP-9 mRNA and, possibly, MMP-2 mRNA.

Role of collagens and perlecan in microvascular stability: exploring the mechanism of capillary vessel damage by snake venom metalloproteinases.

Hemorrhage is a clinically important manifestation of viperid snakebite envenomings, and is induced by snake venom metalloproteinases (SVMPs). Hemorrhagic and non-hemorrhagic SVMPs hydrolyze some basement membrane (BM) and associated extracellular matrix (ECM) proteins. Nevertheless, only hemorrhagic SVMPs are able to disrupt microvessels; the mechanisms behind this functional difference remain largely unknown. We compared the proteolytic activity of the hemorrhagic P-I SVMP BaP1, from the venom of Bothrops asper, and the non-hemorrhagic P-I SVMP leucurolysin-a (leuc-a), from the venom of Bothrops leucurus, on several substrates in vitro and in vivo, focusing on BM proteins. When incubated with Matrigel, a soluble extract of BM, both enzymes hydrolyzed laminin, nidogen and perlecan, albeit BaP1 did it at a faster rate. Type IV collagen was readily digested by BaP1 while leuc-a only induced a slight hydrolysis. Degradation of BM proteins in vivo was studied in mouse gastrocnemius muscle. Western blot analysis of muscle tissue homogenates showed a similar degradation of laminin chains by both enzymes, whereas nidogen was cleaved to a higher extent by BaP1, and perlecan and type IV collagen were readily digested by BaP1 but not by leuc-a. Immunohistochemistry of muscle tissue samples showed a decrease in the immunostaining of type IV collagen after injection of BaP1, but not by leuc-a. Proteomic analysis by LC/MS/MS of exudates collected from injected muscle revealed higher amounts of perlecan, and types VI and XV collagens, in exudates from BaP1-injected tissue. The differences in the hemorrhagic activity of these SVMPs could be explained by their variable ability to degrade key BM and associated ECM substrates in vivo, particularly perlecan and several non-fibrillar collagens, which play a mechanical stabilizing role in microvessel structure. These results underscore the key role played by these ECM components in the mechanical stability of microvessels.

SHh activity and localization is regulated by perlecan.

Proliferation and cell fate determination in the developing embryo are extrinsically regulated by multiple interactions among diverse secreted factors, such as Sonic Hedgehog (SHh), which act in a concentration-dependent manner. The fact that SHh is secreted as a lipid-modified protein suggests the existence of a mechanism to regulate its movement across embryonic fields. We have previously shown that heparan sulfate proteoglycans (HSPGs) are required for SHh binding and signalling. However, it was not determined which specific HSPG was responsible for these functions. Here we evaluated the contribution of perlecan on SHh localization and activity. To understand the mechanism of action of perlecan at the cellular level, we studied the role of perlecan-SHh interaction in SHh activity using both cell culture and biochemical assays. Our findings show that perlecan is a crucial anchor and modulator of SHh activity acting as an extracellular positive regulator of SHh.

Glycosaminoglycans of abdominal skin after massive weight loss in post-bariatric female patients.

BACKGROUND: The number of post-bariatric patients had a significant increase over the last years, and a better understanding of the consequences of massive weight loss on skin is imperative. Despite weight-loss-related changes in collagen and elastin have been reported, less is known about changes in another of the matrix components of the skin, the glycosaminoglycans. The objective of this study is to evaluate abdominal skin glycosaminoglycans concentrations and perlecan and collagen III expression in post-bariatric female patients. METHODS: Skin tissue samples from the abdomen of lean (n = 19) and post-bariatric (n = 24) female patients were compared. Sulfated glycosaminoglycans and hyaluronic acid were extracted, characterized and quantified. Perlecan and collagen III expression was assessed by immunofluorescence. RESULTS: The major glycosaminoglycans found were dermatan sultafe and hyaluronic acid; the others were found in smaller amounts. The skin of the post-bariatric patients had lower concentrations of heparan sulfate (p = 0.002) while hyaluronic acid, dermatan sulfate, and chondroitin sulfate concentrations were similar to the lean women's skin. Post-bariatric skin showed decreased expression of perlecan and increased expression of collagen III. No correlation was found among glycosaminoglycans concentrations and age, body mass index, frequency of pregnancies, or skin types, but it was observed in higher skin heparan sulfate concentrations in post-bariatric patients who had their weights stabilized for over than 24 months (p = 0.000). CONCLUSION: Abdominal skin of post-bariatric women presented decreased heparan sulfate concentrations and perlecan expression and increased expression of collagen III.

Involvement of host cell heparan sulfate proteoglycan in Trypanosoma cruzi amastigote attachment and invasion.

Cell surface glycosaminoglycans (GAGs) play an important role in the attachment and invasion process of a variety of intracellular pathogens. We have previously demonstrated that heparan sulfate proteoglycans (HSPG) mediate the invasion of trypomastigote forms of Trypanosoma cruzi in cardiomyocytes. Herein, we analysed whether GAGs are also implicated in amastigote invasion. Competition assays with soluble GAGs revealed that treatment of T. cruzi amastigotes with heparin and heparan sulfate leads to a reduction in the infection ratio, achieving 82% and 65% inhibition of invasion, respectively. Other sulfated GAGs, such as chondroitin sulfate, dermatan sulfate and keratan sulfate, had no effect on the invasion process. In addition, a significant decrease in infection occurred after interaction of amastigotes with GAG-deficient Chinese Hamster Ovary (CHO) cells, decreasing from 20% and 28% in wild-type CHO cells to 5% and 9% in the mutant cells after 2 h and 4 h of infection, respectively. These findings suggest that amastigote invasion also involves host cell surface heparan sulfate proteoglycans. The knowledge of the mechanism triggered by heparan sulfate-binding T. cruzi proteins may provide new potential candidates for Chagas disease therapy.

SHh activity and localization is regulated by perlecan

Proliferation and cell fate determination in the developing embryo are extrinsically regulated by multiple interactions among diverse secreted factors, such as Sonic Hedgehog (SHh), which act in a concentration-dependent manner. The fact that SHh is secreted as a lipid-modified protein suggests the existence of a mechanism to regulate its movement across embryonic fields. We have previously shown that heparan sulfate proteoglycans (HSPGs) are required for SHh binding and signalling. However, it was not determined which specific HSPG was responsible for these functions. Here we evaluated the contribution of perlecan on SHh localization and activity. To understand the mechanism of action of perlecan at the cellular level, we studied the role of perlecan-SHh interaction in SHh activity using both cell culture and biochemical assays. Our findings show that perlecan is a crucial anchor and modulator of SHh activity acting as an extracellular positive regulator of SHh.

Glial cells modulate heparan sulfate proteoglycan (HSPG) expression by neuronal precursors during early postnatal cerebellar development.

Cerebellum controls motor coordination, balance, eye movement, and has been implicated in memory and addiction. As in other parts of the CNS, correct embryonic and postnatal development of the cerebellum is crucial for adequate performance in the adult. Cellular and molecular defects during cerebellar development can lead to severe phenotypes, such as ataxias and tumors. Knowing how the correct development occurs can shed light into the mechanisms of disease. Heparan sulfate proteoglycans are complex molecules present in every higher eukaryotic cells and changes in their level of expression as well as in their structure lead to drastic functional alterations. This work aimed to investigate changes in heparan sulfate proteoglycans expression during cerebellar development that could unveil control mechanisms. Using real time RT-PCR we evaluated the expression of syndecans, glypicans and modifying enzymes by isolated cerebellar granule cell precursors, and studied the influence of soluble glial factors on the expression of those genes. We evaluated the possible involvement of Runx transcription factors in the response of granule cell precursors to glial factors. Our data show for the first time that cerebellar granule cell precursors express members of the Runx family and that the expression of those genes can also be controlled by glial factors. Our results also show that the expression of all genes studied vary during postnatal development and treatment of precursors with glial factors indicate that the expression of heparan sulfate proteoglycan genes as well as genes encoding heparan sulfate modifying enzymes can be modulated by the microenvironment, reflecting the intricate relations between neuron and glia.

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.

Unique extracellular matrix heparan sulfate from the bivalve Nodipecten nodosus (Linnaeus, 1758) safely inhibits arterial thrombosis after photochemically induced endothelial lesion.

Heparin-like glycans with diverse disaccharide composition and high anticoagulant activity have been described in several families of marine mollusks. The present work focused on the structural characterization of a new heparan sulfate (HS)-like polymer isolated from the mollusk Nodipecten nodosus (Linnaeus, 1758) and on its anticoagulant and antithrombotic properties. Total glycans were extracted from the mollusk and fractionated by ethanol precipitation. The main component (>90%) was identified as HS-like glycosaminoglycan, representing approximately 4.6 mg g(-1) of dry tissue. The mollusk HS resists degradation with heparinase I but is cleaved by nitrous acid. Analysis of the mollusk glycan by one-dimensional (1)H, two-dimensional correlated spectroscopy, and heteronuclear single quantum coherence nuclear magnetic resonance revealed characteristic signals of glucuronic acid and glucosamine residues. Signals corresponding to anomeric protons of nonsulfated, 3- or 2-sulfated glucuronic acid as well as N-sulfated and/or 6-sulfated glucosamine were also observed. The mollusk HS has an anticoagulant activity of 36 IU mg(-1), 5-fold lower than porcine heparin (180 IU mg(-1)), as measured by the activated partial thromboplastin time assay. It also inhibits factor Xa (IC(50) = 0.835 microg ml(-1)) and thrombin (IC(50) = 9.3 microg ml(-1)) in the presence of antithrombin. In vivo assays demonstrated that at the dose of 1 mg kg(-1), the mollusk HS inhibited thrombus growth in photochemically injured arteries. No bleeding effect, factor XIIa-mediated kallikrein activity, or toxic effect on fibroblast cells was induced by the invertebrate HS at the antithrombotic dose.