Search

Placental growth factor (PLGF) and vascular endothelial growth factor-A (VEGF-A) are important regulators of both physiological and pathological vascular remodeling. We previously reported that in monoculture human coronary EC primarily expresses PLGF, while human coronary smooth muscle cells (SMC) primarily express VEGF-A. However, in the vasculature, EC and SMC are in close proximity. Thus, in this study we sought to 1) determine whether this cell-specific expression pattern is maintained when EC and SMC are cocultured, and 2) test the hypothesis that EC and SMC co-regulate the expression of VEGF-A and PLGF. EC and SMC were cultured on either side of a porous Transwell insert and media PLGF and VEGF-A levels were measured by ELISA. We confirmed that the cell type-specific expression of PLGF and VEGF-A we observed in monocultures remains evident in the coculture model. Interestingly, coculture of EC and SMC increased media PLGF relative to EC monoculture, but decreased media VEGF-A compared to SMC monoculture. Coculture conditions also increased VEGFR2 levels on the surface of EC but decreased VEGFR1 levels on the surface of SMC. Inhibition of VEGFR2 tyrosine kinase activity decreased PLGF and increased VEGF in both EC and cocultures but not in SMC monocultures. We conclude that PLGF and VEGF-A exert both paracrine and autocrine regulatory effects on each other and that these effects are mediated by VEGFR2 (in EC) and VEGFR1 (in SMC). Based on these results, we recommend that researchers investigating VEGFR signaling consider the use of coculture models when designing studies.

Atherosclerosis (ASVD) is characterized by the buildup of lipids and fibrous elements in large arteries creating a plaque. As it progresses, apoptotic cells (AC) accumulate forming a necrotic core making the plaque unstable and prone to rupture. Rupture can lead to artery occlusion and moreover to stroke or myocardial infarction (MI). A main contributor to a large necrotic core is the inability of macrophages to clear accumulated AC and as a result undergo secondary necrosis. This clearance process, referred to as efferocytosis, is impaired in advanced atherosclerotic lesions. It has been shown that the process that leads to a thin fibrous cap and a large necrotic core are accelerated by a decrease in glutathione (GSH) availability by a deletion of the modifier subunit of Glutamate Cysteine Ligase (GCLM) in mice. Additionally, it has also been shown that a single nucleotide polymorphism (SNP) in the promoter region of human GCLM (-588 C/T) has an increased risk for MI. Our objective is to identify the role and mechanism of the GCLM SNP during the formation of the necrotic core in atherosclerosis. We hypothesize that proper function of GCLM and GSH is important in macrophages for adequate function and clearance of AC in ASVD. Methods: Human peripheral blood mononuclear cells (PBMCs) were isolated from blood donors, genotyped to test for the presence of SNP, and used for RNA sequencing. Efferocytic index was measured using labelled AC in GCLM WT, Het, and KO murine bone marrow derived macrophages (BMDM) and PBMCs. Percent positive macrophages containing fluorescently labelled AC were quantified for each sample. Cells were collected for total protein, surface receptor expression, and mRNA analysis. Results: Efferocytosis was affected in macrophages with defective GCLM as compared to control. RNA analysis has showed a decrease in efferocytosis receptors such as MerTK, confirmed by cell surface expression. Conclusion: These results suggest that loss of GCLM and GSH in macrophages impairs adequate efferocytosis.

Under physiological and pathological conditions angiogenesis progresses in an idealized redox window. Mice heterozygous for the Glutamate cysteine ligase modifier (GCLm) subunit knockout have augmented ischemic remodeling. During occlusion, the ensuing ischemic milieu and accompanying vascular changes regulate the activity of vascular endothelial receptor 2 (VEGFR2). We hypothesized that the cellular GSH concentration regulates endothelial cell function via altered VEGFR2 signaling. To test this hypothesis, we first performed mouse aortic endothelial cell (MAEC) outgrowth assays on aortic segments dissected from mice homozygous (KO) and heterozygous (Het) for GCLm knockout as well as wildtype control (WT). Outgrowth area in the Het group was significantly higher than WT and KO at day 7 of outgrowth (2.15 fold, n=4, P2 O 2 (100 μM) induced a 5.5 fold (p2 O 2 induced a significant increase in pY416 proto-oncogene c-Src (Src) 9.5 fold (p2 O 2 on VEGFR2. While the VEGFR2 kinase inhibitor SU1948 inhibited activation of VEGFR2, it had no significant effect on the 2-AAPA + H 2 O 2 mediated activation of Src. Combined these data suggest Src mediates the 2-AAPA + H 2 O 2 activation of VEGFR2. Glutathione S-transferase P1 (GSTP1) catalyzes the enzymatic glutathionylation of proteins, The GSTP1 inhibitor TLK199 (50 μM) abrogated the effects of 2-AAPA + H 2 O 2 on VEGFR2 and experiments are underway to determine the glutathionylation status of Src in our system and its effect on the interaction of Src and VEGFR2.

Introduction: In addition to its classic role in redox chemistry, glutathione (GSH) also acts as an allosteric regulator of the calcium-sensing receptor (CaSR). CaSR has been found in a variety of cell types including endothelial cells (ECs), where its activity has been linked to EC adhesion and migration. Although a relationship between GSH and CaSR has been discovered, no link has been established between GSH-CaSR interaction and regulation of angiogenesis. We hypothesize a novel mechanism for GSH regulation of blood vessel development occurs through an extracellular interaction with CaSR as a part of physiological and pathological angiogenesis. Methods: Matrigel tube formation and wound healing assays were used to measure EC activity. RGECO1.2 was used to measure changes in intracellular calcium. Cell surface expression assay was used to measure surface CaSR expression. Cell counts and flow cytometry were used to monitor cell proliferation and cell cycle progression. Endothelial and mesenchymal markers were measured using real-time PCR. Results: Extracellular GSH increased endothelial tube length and branching in tube formation assay, while a selective CaSR inhibitor abrogated these effects. GSH also influenced EC migration as measured by wound healing assay. GSH or a CaSR agonist (R-568) reduced surface CaSR expression, with no changes in intracellular calcium. Extracellular GSH alters EC proliferation and cell cycle progression, coinciding with alterations in endothelial phenotype along the endothelial-mesenchymal spectrum. Conclusion: In addition to its classic antioxidant role, GSH appears to act extracellularly to influence EC activity. GSH caused significant increases in EC branching as measured by tube formation assay. The use of a selective CaSR inhibitor confirms the contribution of CaSR to this effect. Additionally, GSH influenced EC migration in wound healing assays. These results could be explained by GSH-CaSR-mediated alterations in endothelial differentiation. These results suggest a role for GSH in vascular development and angiogenesis, through a ligand interaction with the CaSR G-protein coupled receptor, representing a novel mechanism of action for physiological and pathological blood vessel development.

Purpose Diabetic retinopathy is a vision-threatening complication of diabetes characterized by endothelial injury and vascular dysfunction. The loss of the endothelial glycocalyx, a dynamic layer lining all endothelial cells, contributes to several microvascular pathologies, including an increase in vascular permeability, leukocyte plugging, and capillary occlusion, and may drive the progression of retinopathy. Previously, a significant decrease in glycocalyx thickness has been observed in diabetic retinas. However, the effects of diabetes on specific components of the retinal glycocalyx have not yet been studied. Therefore, the aim of our study was to investigate changes in synthesis, expression, and shedding of retinal glycocalyx components induced by hyperglycemia, which could provide a novel therapeutic target for diabetic retinopathy. Methods Primary rat retinal microvascular endothelial cells (RRMECs) were grown under normal glucose (5 mM) or high-glucose (25 mM) conditions for 6 days. The mRNA and protein levels of the glycocalyx components were examined using qRT-PCR and Western blot analysis, respectively. Further, mass spectrometry was used to analyze protein intensities of core proteins. In addition, the streptozotocin-induced Type 1 diabetic rat model was used to study changes in the expression of the retinal glycocalyx in vivo. The shedding of the glycocalyx was studied in both culture medium and in plasma using Western blot analysis. Results A significant increase in the shedding of syndecan-1 and CD44 was observed both in vitro and in vivo under high-glucose conditions. The mRNA levels of syndecan-3 were significantly lower in the RRMECs grown under high glucose conditions, whereas those of syndecan-1, syndecan-2, syndecan-4, glypican-1, glypican-3, and CD44 were significantly higher. The protein expression of syndecan-3 and glypican-1 in RRMECs was reduced considerably following exposure to high glucose, whereas that of syndecan-1 and CD44 increased significantly. In addition, mass spectrometry data also suggests a significant increase in syndecan-4 and a significant decrease in glypican-3 protein levels with high glucose stimulation. In vivo, our data also suggest a significant decrease in the mRNA transcripts of syndecan-3 and an increase in mRNA levels of glypican-1 and CD44 in the retinas of diabetic rats. The diabetic rats exhibited a significant reduction in the retinal expression of syndecan-3 and CD44. However, the expression of syndecan-1 and glypican-1 increased significantly in the diabetic retina. Conclusions One of the main findings of our study was the considerable diversity of glucose-induced changes in expression and shedding of various components of endothelial glycocalyx, for example, increased endothelial and retinal syndecan-1, but decreased endothelial and retinal syndecan-3. This indicates that the reported decrease in the retinal glycocalyx in diabetes in not a result of a non-specific shedding mechanism. Moreover, mRNA measurements indicated a similar diversity, with increases in endothelial and/or retinal levels of syndecan-1, glypican-1, and CD44, but a decrease for syndecan-3, with these increases in mRNA potentially a compensatory reaction to the overall loss of glycocalyx.

Arterial calcification is an important hallmark of cardiovascular disease and shares many similarities with skeletal mineralisation. The bone-specific protein osteocalcin (OCN) is an established marker of vascular smooth muscle cell (VSMC) osteochondrogenic trans-differentiation and a known regulator of glucose metabolism. However, the role of OCN in controlling arterial calcification is unclear. We hypothesised that OCN regulates calcification in VSMCs and sought to identify the underpinning signalling pathways. Immunohistochemistry revealed OCN co-localisation with VSMC calcification in human calcified carotid artery plaques. Additionally, 3 mM phosphate treatment stimulated OCN mRNA expression in cultured VSMCs (1.72 fold; p

A rapid and efficient method for the detection of hydroxyapatite (HAP) has been developed which shows superiority to existing well-established methods. This fluorescein-bisphosphonate probe is highly selective for HAP over other calcium minerals and is capable of detecting lower levels of calcification in cellular models than either hydrochloric acid-based calcium leaching assays or the Alizarin S stain. The probe has been shown to be effective in both in vitro vascular calcification models and in vitro bone calcification models. Moreover we have demonstrated binding of this probe to vascular calcification in rat aorta and to areas of microcalcification, in human vascular tissue, beyond the resolution of computed tomography in human atherosclerotic plaques. Fluorescein-BP is therefore a highly sensitive and specific imaging probe for the detection of vascular calcification, with the potential to improve not only ex vivo assessments of HAP deposition but also the detection of vascular microcalcification in humans.

The human cardiovascular system is a complex arrangement of specialized structures with distinct functions. The molecular landscape, including the genome, transcriptome and proteome, is pivotal to the biological complexity of both normal and abnormal mammalian processes. Despite our advancing knowledge and understanding of cardiovascular disease (CVD) through the principal use of rodent models, this continues to be an increasing issue in today's world. For instance, as the ageing population increases, so does the incidence of heart valve dysfunction. This may be because of changes in molecular composition and structure of the extracellular matrix, or from the pathological process of vascular calcification in which bone-formation related factors cause ectopic mineralization. However, significant differences between mice and men exist in terms of cardiovascular anatomy, physiology and pathology. In contrast, large animal models can show considerably greater similarity to humans. Furthermore, precise and efficient genome editing techniques enable the generation of tailored models for translational research. These novel systems provide a huge potential for large animal models to investigate the regulatory factors and molecular pathways that contribute to CVD in vivo. In turn, this will help bridge the gap between basic science and clinical applications by facilitating the refinement of therapies for cardiovascular disease.

Placental growth factor (PLGF), a potent stimulator of arteriogenesis, is upregulated during outward arterial remodeling. Increased fluid shear stress (FSS) is a key physiological stimulus for arteriogenesis. However, the role of FSS in regulating PLGF expression is unknown. To test the hypothesis that FSS regulates PLGF expression in vascular cells and to identify the signaling pathways involved, human coronary artery endothelial cells (HCAEC) and human coronary artery smooth muscle cells were cultured on either side of porous Transwell inserts. HCAEC were then exposed to pulsatile FSS of 0.07 Pa (“normal,” mimicking flow through quiescent collaterals), 1.24 Pa (“high,” mimicking increased flow in remodeling collaterals), or 0.00 Pa (“static”) for 2 h. High FSS increased secreted PLGF protein ∼1.4-fold compared with static control ( n = 5, P < 0.01), while normal FSS had no significant effect on PLGF. Similarly, high flow stimulated PLGF mRNA expression nearly twofold in isolated mouse mesenteric arterioles. PLGF knockdown using siRNA revealed that HCAEC were the primary source of PLGF in cocultures ( n = 5, P < 0.01). Both H2O2and nitric oxide production were increased by FSS compared with static control ( n = 5, P < 0.05). NG-nitro-l-arginine methyl ester (100 μM) had no significant effect on the FSS-induced increase in PLGF. In contrast, both catalase (500 U/ml) and diphenyleneiodonium (5 μM) attenuated the effects of FSS on PLGF protein in cocultures. Diphenyleneiodonium also blocked the effect of high flow to upregulate PLGF mRNA in isolated arterioles. Further studies identified NADPH oxidase 4 as a source of reactive oxygen species for this pathway. We conclude that FSS regulates PLGF expression via NADPH oxidase 4 and reactive oxygen species signaling.

Objective PLGF, a VEGF-A related protein, mediates collateral enlargement via monocytes but plays little role in capillary proliferation. In contrast, VEGF-A mediates both collateral enlargement and capillary proliferation. PLGF has been less thoroughly studied than VEGF-A, and questions remain regarding its regulation and function. Therefore, our goal was to characterize the expression of PLGF by vascular cells. We hypothesized that vascular SMC would express more PLGF than EC, since VEGF-A is primarily expressed by non-EC. Methods We compared PLGF and VEGF-A across eight EC and SMC lines, then knocked down PLGF and evaluated cell function. We also assessed the effect of hypoxia on PLGF expression and promoter activity. Results PLGF was most highly expressed in EC, whereas VEGF-A was most highly expressed in SMC. PLGF knockdown did not affect EC number, migration, or tube formation, but reduced monocyte migration toward EC. Monocyte migration was rescued by exogenous PLGF. Hypoxia increased PLGF protein without activating PLGF gene transcription. Conclusions PLGF and VEGF-A have distinct patterns of expression in vascular cells. EC derived PLGF may function primarily in communication between EC and circulating cells. Hypoxia increases EC PLGF expression posttranscriptionally.

Patients with end‐stage renal disease (ESRD) have elevated circulating calcium (Ca) and phosphate (Pi), and exhibit accelerated progression of calcific aortic valve disease (CAVD). We hypothesized that matrix vesicles (MVs) initiate the calcification process in CAVD. Ca induced rat valve interstitial cells (VICs) calcification at 4.5 mM (16.4‐fold; p

Vascular calcification, which is common in the elderly and in patients with atherosclerosis, diabetes and chronic renal disease, increases the risk of cardiovascular morbidity and mortality. It is a complex, active and highly regulated cellular process that resembles physiological bone formation. It has previously been established that pharmacological doses of glucocorticoids facilitate arterial calcification. However, the consequences for vascular calcification of endogenous glucocorticoid elevation have yet to be established. Glucocorticoids (cortisol, corticosterone) are released from the adrenal gland, but can also be generated within cells from 11-keto metabolites of glucocorticoids (cortisone, 11-dehydrocorticosterone [11-DHC]) by the enzyme, 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). In the current study we hypothesized that endogenous glucocorticoids facilitate vascular smooth muscle cell (VSMC) calcification and investigated the receptor-mediated mechanism underpinning this process. In vitro studies revealed increased phosphate-induced calcification in mouse VSMCs following treatment for 7 days with corticosterone (100 nM; 7.98 fold; P, Graphical abstract Image 1

Objective The process of arterial calcification shares many similarities to skeletal mineralisation, and involves the deposition of hydroxyapatite in the arteries. However, the cellular mechanisms responsible have yet to be fully elucidated. Accumulating evidence suggests that aerobic glycolysis (the Warburg effect), plays a critical role in meeting the demand for energy and biosynthetic precursors during proliferation and differentiation in numerous cell types. Therefore we addressed the hypothesis that vascular smooth muscle cell (VSMC) calcification requires aerobic glycolysis to produce energy and the necessary biosynthetic precursors. Methods Calcification of murine aortic VSMCs was induced by 3 mM Pi for 7 days. Calcium deposition was determined using alizarin red staining and a modified o-cresolphthalein method. VSMCs were cultured with the fluorescent glucose analogue 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)−2-Deoxyglucose (2-NBDG) to determine changes in glucose uptake. Gene expression was analysed by qRT-PCR. Results Calcium deposition was significantly increased in VSMCs cultured in 3 mM Pi versus control conditions (124%, p Conclusion Together these data suggest that arterial calcification requires glucose metabolism through a mechanism involving Wnt signalling. Interruption of the glycolysis pathway may therefore represent a novel therapeutic target for clinical intervention.

Arteriogenesis is the process by which mature arteries form from collateral arterioles after upstream arterial stenosis or occlusion. During collateral remodeling, monocytes are recruited to the vessel wall. Placental growth factor (PLGF) is a potent stimulator of arteriogenesis via monocyte recruitment. Although fluid shear stress is thought to be the primary signal for arteriogenesis, its role in regulating PLGF expression is unknown. However, PLGF is increased in collaterals by upstream arterial ligation. Therefore, we hypothesized that PLGF expression is regulated by shear stress. To test this hypothesis, we created an in vitro model of coronary vessels, consisting of human coronary artery endothelial cells (HCAEC) and human coronary artery smooth muscle cells (HCASMC) cocultured on porous Transwell inserts. HCAEC were then exposed to normal (low) shear stress (LSS) (time average 0.7 dyne/cm2), high shear stress (HSS) (time average 12.4 dyne/cm2), or no shear (static) for 2 hours. HSS significantly increased secreted PLGF protein compared to LSS (~25%, n= 5, p < 0.05) as determined by ELISA. Knockdown of PLGF by siRNA in HCAEC significantly reduced secreted PLGF protein while knockdown in HCASMC had no significant effect, suggesting HCAEC are the primary source of PLGF protein (n=4, p

Connective tissue calcifying diseases (CTCs) are characterized by abnormal calcium deposition in connective tissues. CTCs are caused by multiple factors including chronic diseases (Type II diabetes mellitus, chronic kidney disease), the use of pharmaceuticals (e.g. warfarin, glucocorticoids) and inherited rare genetic diseases such as pseudoxanthoma elasticum (PXE), generalized arterial calcification in infancy (GACI) and Keutel syndrome (KTLS). This review explores our current knowledge of these rare inherited CTCs, and highlights the most promising avenues for pharmaceutical intervention. Advancing our understanding of rare inherited forms of CTC is not only essential for the development of therapeutic strategies for patients suffering from these diseases, but also fundamental to delineating the mechanisms underpinning acquired chronic forms of CTC.