Rajiv Janardhanan

Simvastatin reduces venous stenosis formation in a murine hemodialysis vascular access model

Venous neointimal hyperplasia (VNH) is responsible for hemodialysis vascular access malfunction. Here we tested whether VNH formation occurs, in part, due to vascular endothelial growth factor-A (VEGF-A) and matrix metalloproteinase (MMP)-9 gene expression causing adventitial fibroblast transdifferentiation to myofibroblasts (α-SMA-positive cells). These cells have increased proliferative and migratory capacity leading to VNH formation. Simvastatin was used to decrease VEGF-A and MMP-9 gene expression in our murine arteriovenous fistula model created by connecting the right carotid artery to the ipsilateral jugular vein. Compared to fistulae of vehicle-treated mice, the fistulae of simvastatin-treated mice had the expected decrease in VEGF-A and MMP-9 but also showed a significant reduction in MMP-2 expression with a significant decrease in VNH and a significant increase in the mean lumen vessel area. There was an increase in terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, and decreases in α-SMA density, cell proliferation, and HIF-1α and hypoxyprobe staining. This latter result prompted us to determine the effect of simvastatin on fibroblasts subjected to hypoxia in vitro. Simvastatin-treated fibroblasts had a significant decrease in myofibroblast production along with decreased cellular proliferation, migration, and MMP-9 activity but increased caspase 3 activity suggesting increased apoptosis. Thus, simvastatin results in a significant reduction in VNH, with increase in mean lumen vessel area by decreasing VEGF-A/MMP-9 pathway activity.

Increased expression of HIF-1α, VEGF-A and its receptors, MMP-2, TIMP-1, and ADAMTS-1 at the venous stenosis of arteriovenous fistula in a mouse model with renal insufficiency

PURPOSE A mouse model of renal insufficiency with arteriovenous fistula (AVF) and venous stenosis was created. The authors tested the hypothesis that there is increased gene expression of hypoxia-inducible factor-1 alpha (HIF-1alpha); vascular endothelial growth factor-A (VEGF-A) and its receptors (VEGFR-1, -2); matrix metalloproteinase-2 (MMP-2), -9 (MMP-9); tissue inhibitor of metalloproteinase-1, -2 (TIMP-1, -2); and a disintegrin and metalloproteinase thrombospondin-1 (ADAMTS-1) at the venous stenosis. MATERIALS AND METHODS Nineteen male C57BL/6 mice underwent a left nephrectomy and a surgical occlusion of the right upper pole to induce renal function characterized in eight animals. Twenty eight days later, an AVF (n = 11) was created from the right carotid artery to ipsilateral jugular vein, and the mice were killed at day 7 (n = 4) and day 14 (n = 4). The outflow and control veins were removed for gene expression. Three mice were killed at day 28 for histologic analysis. RESULTS The mean serum blood urea nitrogen level remained significantly elevated for 8 weeks when compared with baseline (P < .05). By day seven, there was a significant increase in the expression of HIF-1alpha, VEGF-A, VEGFR-1, VEGFR-2, MMP-2, TIMP-1, and ADAMTS-1 at the outflow vein, with HIF-1alpha and TIMP-1 levels significantly elevated at day 14 (P < .05). By day 28, the venous stenosis was characterized by a thickened vein wall and neointima. CONCLUSIONS A mouse model of renal insufficiency with AVF was developed that had increased expression of HIF-1alpha, VEGF-A, VEGFR-1, VEGFR-2, MMP-2, TIMP-1, and ADAMTS-1 at the outflow vein with venous stenosis by day 28.

Adventitial transduction of lentivirus-shRNA-VEGF-A in arteriovenous fistula reduces venous stenosis formation

Venous neointimal hyperplasia (VNH) causes hemodialysis vascular access failure. Here we tested whether VNH formation occurs in part due to local vessel hypoxia caused by surgical trauma to the vasa vasorum of the outflow vein at the time of arteriovenous fistula placement. Selective targeting of the adventitia of the outflow vein at the time of fistula creation was performed using a lentivirus-delivered small-hairpin RNA that inhibits VEGF-A expression. This resulted in significant increase in mean lumen vessel area, decreased media/adventitia area, and decreased constrictive remodeling with a significant increase in apoptosis (increase in caspase 3 activity and TUNEL staining) accompanied with decreased cellular proliferation and hypoxia-inducible factor-1α at the outflow vein. There was significant decrease in cells staining positive for α-smooth muscle actin (a myofibroblast marker) and VEGFR-1 expression with a decrease in MMP-2 and MMP-9. These results were confirmed in animals that were treated with humanized monoclonal antibody to VEGF-A with similar results. Since hypoxia can cause fibroblast to differentiate into myofibroblasts, we silenced VEGF-A gene expression in fibroblasts and subjected them to hypoxia. This decreased myofibroblast production, cellular proliferation, cell invasion, MMP-2 activity, and increased caspase 3. Thus, VEGF-A reduction at the time of arteriovenous fistula placement results in increased positive vascular remodeling.

Structure-activity study to develop cationic lipid-conjugated haloperidol derivatives as a new class of anticancer therapeutics

Haloperidol (HP), a neuroleptic drug, shows high affinity toward σ receptors (SR). HP and reduced-HP at higher concentration were known to induce apoptosis in SR-overexpressing carcinomas and melanomas. Herein, we report the development of cationic lipid-conjugated haloperidol as a new class of anticancer therapeutics. In comparison to HP, the C-8 carbon chain analogue (HP-C8) showed significantly high, SR-assisted antiproliferative activity against cancer cells via caspase-3-mediated apoptosis and down-regulation of pAkt. Moreover, melanoma tumor aggressiveness in HP-C8-treated mice was significantly lower than that in HP-treated mice. HP-C8 simultaneously reduced Akt-protein level and increased Bax/Bcl-2 ratio in vascular endothelial cells, thereby indicating a possible protein kinase down-regulatory and apoptosis inducing role in tumor-associated vascular cells. In conclusion, we developed σ receptor-targeting cationic lipid-modified HP derivatives as a promising class of anticancer therapeutic that concurrently affects cancer and tumor environment associated angiogenic vascular cells through induction of apoptosis and Akt protein down-regulation.

Effect of Hyperglycemia on Human Monocyte Activation

Our recent study defined the chemokine-induced human monocyte signaling under normoglycemic condition. To explore the hyperglycemia-induced monocyte signaling, we performed adhesion, migration, and transmigration assays on human monocytes obtained from THP-1 cell line in the presence of normal (5 mM) and high (10 and 20 mM) glucose concentrations without chemokines. We observed augmented (P < 0.01) monocyte adhesion to human umbilical vein endothelial cell monolayer at 10 than 5 mM glucose with no further increase at 20-mM glucose concentration (P < 0.07 vs 10 mM; P < 0.01 vs 5 mM). But incremental increases in monocyte migration (P < 0.01), transmigration (P < 0.01), and stress fiber response (P < 0.01) were observed at 10- and 20-mM glucose concentrations in comparison to 5-mM glucose concentrations. We found gradational increase (P < 0.01) in phosphorylation of Akt S473 and glycogen synthase kinase (GSK3βS9) in hyperglycemia (10 and 20 mM) when compared with 5 mM glucose. Furthermore, hyperglycemia (both 10 and 20 mM)-treated monocyte showed up-regulated phosphorylation of p101 and p110γ subunits of PI-3 kinase in comparison to 5 mM glucose. Hyperglycemia-induced monocyte migration was restored to basal levels in the presence of PI-3 kinase inhibitor, LY. These observations imply that modest hyperglycemia per se, as is commonly observed in diabetic individuals, is a potent stimulator of monocyte activity even without chemokines.

Adventitial delivery of lentivirus-shRNA-ADAMTS-1 reduces venous stenosis formation in arteriovenous fistula.

Hemodialysis vascular access can develop venous neointimal hyperplasia (VNH) causing stenosis. Recent clinical and experimental data has demonstrated that there is increased expression of a disintegrin and metalloproteinase thrombospondin motifs-1 (ADAMTS-1) at site of VNH. The experiments outlined in the present paper were designed to test the hypothesis that targeting of the adventitia of the outflow vein of murine arteriovenous fistula (AVF) using a small hairpin RNA that inhibits ADAMTS-1 expression (LV-shRNA-ADAMTS-1) at the time of fistula creation will decrease VNH. At early time points, ADAMTS-1 expression was significantly decreased associated with a reduction in vascular endothelial growth factor-A (VEGF-A) and matrix metalloproteinase-9 (MMP-9) (LV-shRNA-ADAMTS-1 transduced vessels vs. controls). These changes in gene and protein expression resulted in favorable vascular remodeling with a significant increase in mean lumen vessel area, decrease in media/adventitia area, with a significant increase in TUNEL staining accompanied with a decrease in cellular proliferation accompanied with a reduction in CD68 staining. Collectively, these results demonstrate that ADAMTS-1 transduced vessels of the outflow vein of AVF have positive vascular remodeling.

Nanomedicine: pharmacological perspectives

Abstract Rapid developments in the field of nanotechnology are generating enormous interest in prospecting the potential of multifunctional therapeutic nanovectors in the field of personalized medicine. Although the nanomaterials are of same dimensions to many cellular machineries, their interaction with cells, tissues and organs of the body are not well understood. This in turn forms the rationale for a comprehensive study of these nanoplatforms in various disease models in terms of their toxicity, pharmacokinetics, pharmacodynamics, and pharmacogenetics. Such a study will significantly aid in our quest in translating its application from bench to bedside.

Hyperglycemia-Induced Modulation of the Physiognomy and Angiogenic Potential of Fibroblasts Mediated by Matrix Metalloproteinase-2: Implications for Venous Stenosis Formation Associated with Hemodialysis Vascular Access in Diabetic Milieu

Purpose: It is hypothesized that venous stenosis formation associated with hemodialysis vascular-access failure is caused by hypoxia-mediated fibroblast-to-myofibroblast differentiation accompanied by proliferation and migration, and that diabetic patients have worse clinical outcomes. The aim of this study was to determine the functional and gene expression outcomes of matrix metalloproteinase-2 (Mmp-2) silencing in fibroblasts cultured under hyperglycemia and euglycemia with hypoxic and normoxic stimuli. Materials and Methods: AKR-2B fibroblasts were stably transduced using lentivirus-mediated shRNA-Mmp-2 or scrambled controls and subjected to hypoxia or normoxia under hyperglycemic or euglycemic conditions for 24 and 72 h. Gene expression of vascular endothelial growth factor-A (Vegf-A), Vegfr-1, Mmp-2, Mmp-9 and tissue inhibitors of matrix metalloproteinases (Timps) were determined by RT-PCR. Collagen I and IV secretion and cellular proliferation and migration were determined. Results: Under hyperglycemic conditions, there is a significant reduction in the average gene expression of Vegf-A and Mmp-9, with an increase in Timp-1 at 24 h of hypoxia (p < 0.05) in Mmp-2-silenced fibroblasts when compared to controls. In addition, there is a decrease in collagen I and IV secretion and cellular migration. The euglycemic cells were able to reverse these findings. Conclusion: These findings demonstrate the rationale for using anti-Mmp-2 therapy in dialysis patients with hemodialysis vascular access in helping to reduce stenosis formation.

Letter to the EditorThe Authors Reply

We would like to thank Drs. Eroglu and Kocyigit for their comment on possible use of colchicine on arteriovenous fistula maturation via neointimal hyperplasia blockage.1 We used simvastatin because it is a pleiotropic molecule capable of modulating the gene expression of vascular endothelial growth factor-A and matrix metalloproteinase-9. In our studies, simvastatin treated vessels had improved vascular remodeling in a experimental murine model of arteriovenous fistula (AVF) with chronic kidney disease using a clinical dose 2. As pointed out by Eroglu and Kocyigit, simvastatin is associated with rhabdomyolysis, hepato-toxicity, and uremia induced cardiomyopathy 3. Taking this into consideration, we have begun to consider local delivery of simvastatin to the vessel wall using nanoparticles or alginate gel wraps to decrease these side effects. Colchicine treatment for six months has been shown recently to decrease in stent restenosis in patients with coronary disease treated with bare metal stents 4. This is felt to be because of its ability to inhibit microtubule formation as well as an anti-inflammatory component 5. Furthermore, it is interesting that a trial using colchicine was negative in patients undergoing angioplasty of coronary arteries while a separate trial in patients undergoing stent placement was positive 4, 6. The mechanism for this observation may be due to elastic recoil, which occurs in the artery after angioplasty and also in the vein. In conclusion, we think colchicine is a plausible therapy, which may reduce venous neointimal hyperplasia in patients with hemodialysis vascular access, and further studies using this molecule need to be performed to determine its efficacy.

Response to the Letter entitled “The possible effect of colchicine on arteriovenous fistula maturation via neointimal hyperplasia blockage”

We would like to thank Drs. Eroglu and Kocyigit for their comment on possible use of colchicine on arteriovenous fistula maturation via neointimal hyperplasia blockage.1 We used simvastatin because it is a pleiotropic molecule capable of modulating the gene expression of vascular endothelial growth factor-A and matrix metalloproteinase-9. In our studies, simvastatin treated vessels had improved vascular remodeling in a experimental murine model of arteriovenous fistula (AVF) with chronic kidney disease using a clinical dose 2. As pointed out by Eroglu and Kocyigit, simvastatin is associated with rhabdomyolysis, hepato-toxicity, and uremia induced cardiomyopathy 3. Taking this into consideration, we have begun to consider local delivery of simvastatin to the vessel wall using nanoparticles or alginate gel wraps to decrease these side effects. Colchicine treatment for six months has been shown recently to decrease in stent restenosis in patients with coronary disease treated with bare metal stents 4. This is felt to be because of its ability to inhibit microtubule formation as well as an anti-inflammatory component 5. Furthermore, it is interesting that a trial using colchicine was negative in patients undergoing angioplasty of coronary arteries while a separate trial in patients undergoing stent placement was positive 4, 6. The mechanism for this observation may be due to elastic recoil, which occurs in the artery after angioplasty and also in the vein. In conclusion, we think colchicine is a plausible therapy, which may reduce venous neointimal hyperplasia in patients with hemodialysis vascular access, and further studies using this molecule need to be performed to determine its efficacy.