Kalisha D. O'Neill

Annexin-Mediated Matrix Vesicle Calcification in Vascular Smooth Muscle Cells

In bone, osteoblasts and chondrocytes synthesize matrix vesicles (MVs) that interact with collagen to initiate calcification. MVs have been identified in human calcified arteries but are poorly characterized. The objective of this study is to determine the role of annexins and fetuin‐A in MV formation and activity during calcification in bovine vascular smooth muscle cells (BVSMCs). BVSMCs were treated with control or calcification (high phosphorus) media, and cellular MVs were isolated by collagenase digestion and secreted MVs were isolated from cultured media by ultracentrifugation. The results showed that alkaline phosphatase (ALP) activity was significantly increased in MVs from calcified BVSMCs compared with noncalcified BVSMCs, as was annexin II and VI content and 45Ca uptake. We also determined that MVs from calcifying BVSMCs could mineralize type I collagen but not type II collagen in the absence of cells in a dose‐ and time‐dependent manner. Blockade of annexin calcium channel activity by K201 significantly decreased ALP activity and reduced the ability of the MVs to subsequently calcify on collagen, whether the K201 was added during or after MV formation. Furthermore, cellular MVs had significantly increased ability to calcify on collagen compared with secreted MVs, likely because of their increased ALP activity and annexin II content but low fetuin‐A content. In conclusion, our results suggest that mineralization in VSMCs requires both active MVs and an interaction of the MVs with type I collagen, and both steps require annexin activity.

Decreased MicroRNA Is Involved in the Vascular Remodeling Abnormalities in Chronic Kidney Disease (CKD)

Patients with CKD have abnormal vascular remodeling that is a risk factor for cardiovascular disease. MicroRNAs (miRNAs) control mRNA expression intracellularly and are secreted into the circulation; three miRNAs (miR-125b, miR-145 and miR-155) are known to alter vascular smooth muscle cell (VSMC) proliferation and differentiation. We measured these vascular miRNAs in blood from 90 patients with CKD and found decreased circulating levels with progressive loss of eGFR by multivariate analyses. Expression of these vascular miRNAs miR-125b, miR-145, and miR-155 was decreased in the thoracic aorta in CKD rats compared to normal rats, with concordant changes in target genes of RUNX2, angiotensin II type I receptor (AT1R), and myocardin. Furthermore, the expression of miR-155 was negatively correlated with the quantity of calcification in the aorta, a process known to be preceded by vascular de-differentiation in these animals. We then examined the mechanisms of miRNA regulation in primary VSMC and found decreased expression of miR-125b, 145, and 155 in VSMC from rats with CKD compared to normal littermates but no alteration in DROSHA or DICER, indicating that the low levels of expression is not due to altered intracellular processing. Finally, overexpression of miR-155 in VSMC from CKD rats inhibited AT1R expression and decreased cellular proliferation supporting a direct effect of miR-155 on VSMC. In conclusion, we have found ex vivo and in vitro evidence for decreased expression of these vascular miRNA in CKD, suggesting that alterations in miRNAs may lead to the synthetic state of VSMC found in CKD. The decreased levels in the circulation may reflect decreased vascular release but more studies are needed to confirm this relationship.

Activation of arterial matrix metalloproteinases leads to vascular calcification in chronic kidney disease

Background: The objective of the current study was to determine if altered regulation of matrix metalloproteinases (MMPs) may predispose to extracellular matrix degradation, facilitating arterial calcification in chronic kidney disease (CKD) using a progressive model of CKD-MBD, the Cy/+ rat. Methods: Sera were collected from normal or CKD rats at various times and MMP-2 and MMP-9 levels determined by ELISA or zymography. Aorta tissue was harvested at sacrifice for RT-PCR and immunostaining. Calcification of aorta rings was assessed with MMP inhibitors. Results: There was an increase in MMP-2, MMP-9, TIMP-1, and RUNX-2 expression in the aorta with progressive CKD, and increased MMP-2 activity in the serum. Immunostaining revealed increased expression of MMP-2 and MMP-9 in areas of aorta calcification. There was also an upregulation of MMP-2 and MMP-9 in vascular smooth muscle cells (VSMC) from CKD rats. MMP inhibitors decreased calcification of aorta rings from normal and CKD rats. High phosphorus increased MMP-2 and MMP-9 expressions in VSMC from normal rats but not from CKD rats. Conclusion: MMP-2 and MMP-9 expression and activity are increased with progressive CKD, and blockade of MMP activity can inhibit arterial calcification. These data suggest degradation of the extracellular matrix is a critical step in the pathogenesis of arterial calcification in CKD.

Verapamil inhibits calcification and matrix vesicle activity of bovine vascular smooth muscle cells

Calcium channel activity in vascular smooth muscle cells is a critical component during vascular calcification and formation of matrix vesicles. Here, we examined whether the blockade of L-type calcium channels inhibits these functions. Bovine vascular smooth muscle cells or rat aorta organ cultures were incubated in media known to promote calcification and treated with the L-type calcium channel inhibitors verapamil, nifedipine, or nimodipine. The phenylalkylamine, verapamil, significantly decreased calcification of the vascular smooth muscle cells and rat aorta, in a dose-dependent manner, whereas the dihydropyridines, nifedipine and nimodipine, had no effect. Furthermore, verapamil, but not nifedipine, significantly decreased the alkaline phosphatase activity of bovine vascular smooth muscle cells. Verapamil pretreatment of the cells also inhibited matrix vesicle alkaline phosphatase activity and reduced the ability of these matrix vesicles to subsequently calcify on a type I collagen extracellular matrix scaffold. As L-type channels are blocked by verapamil and dihydropyridines, we suggest that verapamil inhibits vascular smooth muscle mineralization and matrix vesicle activity by mechanisms other than the simple blockade of this calcium channel activity.

Transglutaminase 2 Accelerates Vascular Calcification in Chronic Kidney Disease

Background: Transglutaminase 2 (TGM2) is a calcium-dependent enzyme that can cross-link nearly all extracellular matrix (ECM) proteins and can facilitate cell-ECM interaction through integrins. Given the importance of the ECM in vascular calcification we tested the hypothesis that increased TGM2 activity may accelerate vascular calcification in chronic kidney disease (CKD). Methods: We utilized thoracic aortas and vascular smooth muscle cells (VSMC) from the Cy/+ rat, a model of progressive CKD that develops arterial calcification on a normal phosphorus diet, compared to normal rats. Results: VSMC isolated from CKD rats had increased expression and activity of TGM2 compared to cells from normal rats. The increased calcification and expression of alkaline phosphatase activity observed in VSMC from CKD rats compared to normal was inhibited in a dose-dependent manner with the TGM inhibitors cystamine and Z006. Matrix vesicles (MV) from CKD rat VSMC also had increased TGM2 expression and the calcification of MV on type I collagen could be inhibited with cystamine and accelerated by exogenous cross-linking of fibronectin or type I collagen with TGM2. Finally, the calcification of aorta rings from CKD rats in ex vivo cultures was inhibited with TGM2 inhibitor. Conclusion: These data demonstrate a role of TGM2 in the pathogenesis of vascular calcification in CKD through enhancement of MV-ECM calcification.

RhoA/Rho kinase (ROCK) alters fetuin-A uptake and regulates calcification in bovine vascular smooth muscle cells (BVSMC)

RhoA/Rho kinases (ROCK) play a critical role in vascular smooth muscle cell (VSMC) actin cytoskeleton organization, differentiation, and function and are implicated in the pathogenesis of cardiovascular disease. We have previously determined that an important step in the regulation of calcification is fetuin-A endocytosis, a process that is dependent on changes in the cytoskeleton, which, in turn, is known to be affected by the RhoA/ROCK signaling pathway. In the present study, bovine VSMC (BVSMC) were treated with the ROCK inhibitor Y-27632 or transfected with ROCK small interfering (si) RNA to knock down ROCK expression. Both conditions resulted in reduced actin stress fibers and increased Cy5-labeled fetuin-A uptake. Inhibition of ROCK by Y-27632 or siRNA also significantly increased BVSMC alkaline phosphatase (ALP) activity and calcification of BVSMC and rat aorta organ cultures. Cells were then incubated in calcification media in the presence or absence of Y-27632 and matrix vesicles (MV) isolated by collagenase digestion. These MV, isolated from BVSMC incubated with Y-27632, had increased ALP activity and increased ability of MV to subsequently calcify collagen by 66%. In contrast, activation of RhoA, which is upstream of ROCK, by transfecting plasmids encoding the dominant active Rho GTPase mutant (Rho-L63) led to decreased fetuin-A uptake and reduced calcification in BVSMC. These results demonstrate that the RhoA/ROCK signaling pathway is an important negative regulator of vascular calcification.

Adipocyte induced arterial calcification is prevented with sodium thiosulfate.

BACKGROUND Calcification can occur in fat in multiple clinical conditions including in the dermis, breasts and in the abdomen in calciphylaxis. All of these are more common in patients with advanced kidney disease. Clinically, hyperphosphatemia and obesity are risk factors. Thus we tested the hypothesis that adipocytes can calcify in the presence of elevated phosphorus and/or that adipocytes exposed to phosphorus can induce vascular smooth muscle cell (VSMC) calcification. METHODS 3T3-L1 preadipocytes were induced into mature adipocytes and then treated with media containing high phosphorus. Calcification was assessed biochemically and PCR performed to determine the expression of genes for osteoblast and adipocyte differentiation. Adipocytes were also co-cultured with bovine VSMC to determine paracrine effects, and the efficacy of sodium thiosulfate was determined. RESULTS The results demonstrated that high phosphorus induced the calcification of differentiated adipocytes with increased expression of osteopontin, the osteoblast transcription factor Runx2 and decreased expression of adipocyte transcription factors peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT-enhancer-binding protein α (CEBPα), indicating that high phosphorus led to a phenotypic switch of adipocytes to an osteoblast like phenotype. Sodium thiosulfate, dose dependently decreased adipocyte calcification and inhibited adipocyte induced increase of VSMC calcification. Co-culture studies demonstrated that adipocytes facilitated VSMC calcification partially mediated by changes of secretion of leptin and vascular endothelial growth factor (VEGF) from adipocytes. CONCLUSION High phosphorus induced calcification of mature adipocytes, and adipocytes exposed to elevated phosphorus can induce calcification of VSMC in a paracrine manner. Sodium thiosulfate inhibited this calcification and decreased the secretin of leptin and VEGF from adipocytes. These results suggest that adipocyte exposure to elevated phosphorus may be a pathogenic factor in calcification observed in the skin in calciphylaxis and other diseases.

Matrix vesicles induce calcification of recipient vascular smooth muscle cells through multiple signaling pathways

In patients with chronic kidney and end-stage renal diseases, the major risk factor for progression of arterial calcification is the presence of existing (baseline) calcification. Here, we tested whether calcification of arteries is extended from calcified vascular smooth muscle cells (VSMCs) to adjacent normal cells by matrix vesicle-induced alteration of cell signaling. Matrix vesicles isolated from VSMC of rats with chronic kidney disease were co-cultured with VSMCs from normal littermates. Endocytosis of vesicles by recipient cells was confirmed by confocal microscopy. The addition of cellular matrix vesicles with characteristics of exosomes and low fetuin-A content enhanced the calcification of recipient VSMC. Further, only cellular-derived matrix vesicles induced an increase in intracellular calcium ion concentration, NOX1 (NADPH oxidase) and the anti-oxidant superoxide dismutase-2 in recipient normal VSMC. The increase in intracellular calcium ion concentration was due to release from endoplasmic reticulum and partially attributed to the activation of both NOX1 and mitogen-activated protein kinase (MEK1 and Erk1/2) signaling, since inhibiting both pathways blocked the increase in intracellular calcium ion in recipient VSMC. In contrast, matrix vesicles isolated from the media had no effect on the intracellular calcium ion concentration or MEK1 signaling, and did not induce calcification. However, media matrix vesicles did increase Erk1/2, although not to the level of cellular matrix vesicles, and NOX1 expression. Blockade of NOX activity further inhibited the cellular matrix vesicle-induced accelerated calcification of recipient VSMC, suggesting a potential therapeutic role of such inhibition. Thus, addition of cellular-derived matrix vesicles from calcifying VSMC can accelerate calcification by inducing cell signaling changes and phenotypic alteration of recipient VSMC.

Hemodialysis does not alter in vitro hepatic CYP3A4 and CYP2D6 metabolic activity in uremic serum

There is a paucity of studies evaluating the change in liver metabolism in subjects receiving hemodialysis. The purpose of this study was to compare the effect of uremic toxins on hepatic cytochrome P450 (CYP)3A4 and CYP2D6 metabolism before and after a 4-hour hemodialysis session. Midazolam and dextromethorphan were incubated with uremic serum collected from subjects before and after the 4-hour hemodialysis session. Analysis and quantification of the 1′-OH-midazolam and 4-OH-midazolam and dextrorphan metabolites were performed by high-pressure liquid chromatography/mass spectrometry. Statistical analysis using the Student’s t-test (paired) was used to compare the amount of metabolite formed. The mean amount of 1′-OH-midazolam, 4-OH-midazolam, and dextrorphan metabolites formed before and after hemodialysis did not significantly differ. There was no significant difference in CYP3A4 and CYP2D6 metabolic activity in uremic serum before and after hemodialysis.