C. C. Kartha

1,25-dihydroxyvitamin D3 receptor is upregulated in aortic smooth muscle cells during hypervitaminosis D

Several studies have demonstrated that excess of vitamin D3 is toxic particularly to vascular tissues. A notable pathological feature is arterial calcification. The nature of the toxic metabolite in hypervitaminosis D and the pathogenesis of arterial calcification are not clearly understood. The present study was undertaken to explore whether arterial calcification is a sequel of increased calcium uptake by arterial smooth muscle mediated by up regulation of vitamin D receptor in the cells in response to elevated circulating levels of vitamin D3 in serum. The experimental study was performed in 20 New Zealand white female rabbits aged 6 months. Animals in the test group were injected 10,000 IU of cholecalciferol intramuscularly twice a week for one month. Six control animals were given intra-muscular injections of plain cottonseed oil. Animals were sacrificed and aortas were examined for pathological lesions, 1,25-dihyroxyvitamin D3 (1,25(OH)2 D3) receptor levels and 45Ca uptake in smooth muscle cells. Serum samples collected at intervals were assayed for levels of 25-OH-D3 and calcium. The results showed that in animals given injections of cholecalciferol, serum levels of 25-OH-D3 were elevated. In four of these animals calcification and aneurysmal changes were seen in the aorta. Histological lesions comprised of fragmentation of elastic fibers as well as extensive loss of elastic layers. 1,25(OH)2 D3 receptor levels were up regulated and 45Ca uptake enhanced in aortas of animals which were given excessive vitamin D3. The evidences gathered suggest that excess vitamin D is arteriotoxic and that the vitamin induces arterial calcification through up regulation of 1,25(OH)2D3 receptor and increased calcium uptake in smooth muscle cells of the arteries.

Quercetin attenuates Monocyte Chemoattractant Protein-1 gene expression in glucose primed aortic endothelial cells through NF-κB and AP-1

Monocyte Chemoattractant Protein-1 (MCP-1) is involved in the diapedesis of blood monocytes into the arterial intima, an early critical event in atherogenesis. Modulating MCP-1 expression can be a key strategy to decrease the risk for atherosclerosis in diabetes. We hypothesized that quercetin, an anti-inflammatory molecule could modulate high glucose concentration (HG) induced MCP-1 expression in aortic endothelial cells in vitro because of its regulatory effects on Activator Protein-1 (AP-1) and Nuclear Factor-kappaB (NF-kappaB). Rat aortic endothelial cells (RAECs) were exposed to HG in the presence or absence of quercetin. Quercetin attenuated HG induced MCP-1 mRNA (42%) and protein synthesis (45%) when estimated using real-time reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay respectively. Western blot analysis found quercetin to maintain cytosolic p65 protein levels to that seen in control. Quercetin was found to attenuate HG induced increased NF-kappaB and AP-1 DNA binding activity in electrophoretic mobility shift assay. Immunofluorescence studies revealed quercetin to prevent HG induced nuclear localization of p65 and c-jun. Quercetin was also found to decrease HG induced activation of NF-kappaB (71%+/-14%), AP-1 (69%+/-24%) and MCP-1 promoter (79%+/-25%) in EA.hy926 cells when analyzed using luciferase reporter assay. We conclude that quercetin attenuates MCP-1 expression in HG treated RAECs, probably by regulating both NF-kappaB and AP-1 pathways. The findings provide new insights into HG induced MCP-1 gene regulation in aortic endothelial cells and the potential of quercetin in abating the risk for atherosclerosis in diabetes.

Effects of epidermal growth factor on proliferation and migration of cardiosphere-derived cells expanded from adult human heart

Recent studies have provided evidence that the human heart has an endogenous reserve of cardiac stem cells (CSCs) that can be activated to reconstitute the dead myocardium. Current efforts are now directed towards the identification of factors favoring the growth and expansion of the CSC pool in the heart. Accordingly, in the present study, effects of different growth factors on cardiosphere-derived cells (CDCs), expanded from atrial biopsies from patients undergoing elective coronary artery bypass surgery, were analyzed. CSCs appear to respond to epidermal growth factor (EGF) more efficiently than other widely used growth factors such as vascular endothelial growth factor, insulin-like growth factor, basic fibroblast growth factor, hepatocyte growth factor, transforming growth factor, and platelet-derived growth factor. EGF significantly promoted cardiosphere formation (p < 0.05) and proliferation (p < 0.005), migration (p < 0.0005), and wound healing (p < 0.005) activities of CDCs in comparison to the other growth factors studied. Pretreatment with EGF enhanced the expression of cardiac markers cTN1+ and MHC+ in CDCs in comparison to untreated controls.

Curcumin Attenuates Glucose-Induced Monocyte Chemoattractant Protein-1 Synthesis in Aortic Endothelial Cells by Modulating the Nuclear Factor-ĸB Pathway

Background/Aims: High glucose (HG) induces monocyte chemoattractant protein-1 (MCP-1) synthesis in endothelial cells through nuclear factor ĸB (NFĸB). We investigated whether curcumin, losartan and sodium salicylate (NaSal) attenuate HG-induced MCP-1 synthesis in rat aortic endothelial cells (RAECs) and explored the mechanism of action. Methods: RAECs were stimulated with HG (25 mmol/l) for 24 h in the presence or absence of curcumin, losartan, NaSal or NFĸB inhibitor, Bay 11-0782. The MCP-1 protein and mRNA levels were determined by enzyme-linked immunosorbent assay and real-time reverse transcriptase-polymerase chain reaction, respectively. Nuclear translocation of NFĸB subunit p65 and NFĸB DNA-binding activity was studied using confocal microscopy and electrophoretic mobility shift assay, respectively. Results: A significant increase in the synthesis of MCP-1 protein and mRNA (2-fold) was observed in HG-primed RAECs compared to control glucose (5.5 mmol/l). Curcumin (30 µmol/l) significantly decreased HG-induced MCP-1 protein (74%) and mRNA (53%) synthesis. There was no inhibition of HG-induced MCP-1 protein secretion by losartan and NaSal. In HG-stimulated RAECs, curcumin attenuated the nuclear translocation of p65 and decreased the NFĸB DNA-binding activity. Conclusion: Curcumin blocks HG-induced MCP-1 synthesis in RAECs partly via the NFĸB pathway.

Endocardial endothelial celsl stimulate proliferation and collagen synthesis of cardiac fibroblasts

Given that vascular endothelial cells play an important role in the modulation of vascular structure and function, we hypothesized that endocardial endothelial cells (EECs) may have a modulator role in regulating the cardiac interstitial cells. Endocardial endothelial cells were isolated from freshly collected pig hearts and cardiac fibroblasts were isolated from 3- to 4-d-old Wistar rats. Fibroblasts were cultured in the presence or absence of conditioned medium from EECs. Proliferation of cardiac fibroblasts was measured by the incorporation of [3H]-Thymidine and collagen synthesis was assayed by the incorporation of [3H]-proline. To determine the involvement of signaling mediators, in separate experiments, cardiac fibroblasts were incubated with BQ123 (selective ETA receptor antagonist), PD142893 (nonselective ETA/ETB receptor antagonist), Bis-indolylmaleimide (PKC inhibitor), PD 098059 (MEK inhibitor), or neutralizing anti-transforming growth factor (TGF)-β-antibody. Endocardial endothelium-derived factors endothelin (ET)-1, TGF-β, and Angiotensin (Ang)-II in the conditioned medium were assayed by enzyme-linked immunosorbent assay using commercially available kits. We report here evidence that suggest that endocardial endothelial cells stimulate both proliferation and collagen synthesis of cardiac fibroblasts. The response seems to be mediated by endothelin through its ETA receptor.Our results also indicate that protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) pathways are essential for the EEC-induced proliferation of cardiac fibroblasts.

Synthetic hydrogel microspheres as substrata for cell adhesion and growth

SummaryCross-linked poly(methyl methacrylate) (PMMA) microspheres were subjected to alkaline hydrolysis to obtain hydrophilic microspheres having carboxyl residues distributed throughout the matrix. These microspheres were found to support the growth of human skin fibroblasts and human heart and lung cells. Further, fibroblasts grown on them were found to be comparable with those grown on the commercial tissue culture plate with respect to [14C]amino acid uptake and incorporation into proteins. The hydrolyzed PMMA microspheres may find application as a microcarrier for cell culture.

Proliferation of murine c-kitpos cardiac stem cells stimulated with IGF-1 is associated with Akt-1 mediated phosphorylation and nuclear export of FoxO3a and its effect on downstream cell cycle regulators

Abstract Insulin-like growth factor-1 (IGF-1) is known to promote proliferation in many cell types including c-kitpos cardiac stem cells (CSCs). Downstream signaling pathways of IGF-1 induced CSC proliferation have not been investigated. An important downstream target of IGF-1/Akt-1 signaling is FoxO3a, a key negative regulator of cell-cycle progression. We studied the effect of IGF-1 on proliferation of c-kitpos murine CSCs and found that IGF-1-mediated cell proliferation is associated with FoxO3a phosphorylation and inactivation of its transcriptional activity. PI3 inhibitors LY294002 and Wortmannin abolished the effect of IGF-1 on FoxO3a phosphorylation indicating that FoxO3a phosphorylation is mediated by PI3/Akt-1 pathway. In cells with FoxO3a translocation to the cytoplasm, there is decreased expression of cell-cycle inhibitors such as p27kip1 and p57kip2 and increased expression of CyclinD1. Our study provides evidence that IGF-1 induced CSC proliferation could be the result of FoxO3a inactivation and its downstream effect on cell-cycle regulators.

Immortalization and Characterization of Porcine Ventricular Endocardial Endothelial Cells

Endocardial endothelial cells (EECs), which form the inner lining of the cavities of the heart, are a distinct cell population whose dysfunction can be critical in pathological conditions of heart. Insights into the role and organization of these cells in pathological states of the heart are limited mainly due to a dearth of experimental models. To date no endocardial endothelial cell line is available. The authors attempted to immortalize porcine ventricular EECs by transfecting the cells with human telomerase reverse transcriptase (hTERT). EECs immortalized by ectopic expression of hTERT exhibit phenotypic and functional characteristics similar to primary EECs. The EE cell line could be useful for the study of mechanisms involved in the interaction of EECs with the underlying myocardium and cardiac interstitium and as useful tools in understanding their role in diseased states of heart.

Effect of Apoptosis-Inducing Antitumor Agents on Endocardial Endothelial Cells

Chemotherapy is one of the common treatment modalities for cancer. Some of the antineoplastic drugs have, however, been found to be toxic for vascular endothelium, resulting in complications such as endothelial dysfunction, thromboembolism, heart failure, and cardiomyopathy. In this study, we investigated the cytotoxic effect of widely used antitumor agents doxorubicin, camptothecin, and thapsigargin on primary and immortalized porcine endocardial endothelial cells and compared with the effects of these agents on human umbilical vein endothelial cells, human aortic endothelial cells, and EA.hy926 cells. Our study revealed that endocardial endothelial cells are relatively resistant to apoptosis induced by these drugs. Interestingly, our study indicates that response to antitumor agents greatly differs depending on the site of origin of endothelial cells. Doxorubicin, camptothecin, and thapsigargin induce mitochondrial-dependent cell death following loss of mitochondrial membrane potential (MMP) in vascular endothelial cells, with subsequent increase in sub-G0 population. In endocardial endothelial cells, there was no MMP loss; and only cell cycle arrest either at G1 or S phases was observed when the cells were treated with doxorubicin, camptothecin, and thapsigargin.

Isolation of ckit-Positive Cardiosphere-Forming Cells from Human Atrial Biopsy

There is increasing interest in developing cell-based therapies to regenerate functional muscle and blood vessels in infarcted dysfunctional myocardium, using stem cells resident in the adult heart. The objective of our study was to identify an easy and cost-effective method for the isolation and expansion of human adult cardiac-resident stem cells. The cells were isolated from right atrial biopsy samples obtained from patients with ischemic heart disease, who were undergoing coronary artery bypass grafting. Two different isolation methods, enzymatic and nonenzymatic, were employed. The cell yield and cluster formation were not significantly different with either of the techniques used for cell isolation. The nonenzymatic method is recommended because of its simplicity and lower cost compared to the enzymatic method.