Biomedical subjects

Ankur Chandra

Publications and source records attributed to Ankur Chandra.

5 recordsLinked to original sources

PubMed2006

A novel fluorescence-based cellular permeability assay.

Vascular permeability is a pathologic process in many disease states ranging from metastatic progression of malignancies to ischemia-reperfusion injury. In order to more precisely study tissue, and more specifically cell layer permeability, our goal was to create a fluorescence-based assay which could quantify permeability without radioactivity or electrical impedance measurements. Human aortic endothelial cells were grown in monolayer culture on Costar-Transwell clear polyester membrane 6-well cell culture inserts. After monolayer integrity was confirmed, vascular endothelial growth factor (VEGF(165)) at varying concentrations with a fixed concentration of yellow-green fluorescent 0.04 microm carboxylate-modified FluoSpheres microspheres were placed in the luminal chamber and incubated for 24 h. When stimulated with VEGF(165) at 20, 40, 80, and 100 ng/ml, this assay system was able to detect increases in trans-layer flux of 8.2+/-2.4%, 16.0+/-3.7%, 41.5+/-4.9%, and 58.6+/-10.1% for each concentration, respectively. This represents the first fluorescence-based permeability assay with the sensitivity to detect changes in the permeability of a cell layer to fluid flux independent of protein flux; as well as being simpler and safer than previous radioactive-and impedance-based permeability assays. With the application of this in vitro assay to a variety of pathologic conditions, both the dynamics and physiology relating to cellular permeability can be more fully investigated.

Biophysics↗

PubMed2005

VEGF inhibits PDGF-stimulated calcium signaling independent of phospholipase C and protein kinase C.

INTRODUCTION: Despite advances in both open and endovascular techniques for treatment of arterial occlusive disease, restenosis because of neointimal hyperplasia continues to be a major cause of graft failure and restenosis. This phenomenon has been attributed to vascular smooth muscle cell (VSMC) activation by several potent mitogens including platelet derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) released at the site of injury. PDGF is known to stimulate calcium influx in VSMC that has been shown to be critical for VSMC migration and proliferation. We have previously shown that VEGF inhibits PDGF-stimulated VSMC proliferation. The objective of this set of experiments was to investigate whether VEGF modulated PDGF-stimulated Ca2+ influx in VSMC. MATERIALS AND METHODS: Primary cultured human aortic SMC were grown to subconfluency and assigned to the following groups: no stimulation, stimulation with PDGF-BB (20 ng/ml), stimulation with VEGF165 (40 ng/ml), or a combination of PDGF-BB + VEGF165. Ca2+ influx was measured using a Fura-2 fluorescence assay. The intracellular Ca2+ fraction was assayed with the Fura-2 assay by using Ca2+-free media. Phospholipase Cgamma1 (PLCgamma1), protein kinase C (PKC), and Akt phosphorylation was assessed with standard immunoblotting techniques at 1, 5, and 10 min time points. Ca2+-calmodulin kinase II (CaMKII) activity was extrapolated from the phosphorylation of Phospholamban B (PLB), a well-known protein substrate, at 1, 5, and 10 min time points. RESULTS: PDGF stimulation resulted in a 328 +/- 9 nm total calcium influx in VSMC. The combination of VEGF + PDGF resulted in a 273 +/- 21 nm total calcium influx, an amount significantly less than with PDGF alone (P < 0.04). PDGF stimulation resulted in a 72 +/- 35 nm intracellular calcium release. The addition of VEGF to PDGF resulted in an intracellular calcium release of only 15 +/- 11 nm, a significant decrease compared to PDGF alone (P < 0.01). The phosphorylation of PLCgamma1, PKC, and Akt was equivalent at 1, 5, and 10 min between the PDGF and the PDGF + VEGF treatment groups. There was an increase in CaMKII activity at 1 and 5 min time points in both the PDGF and PDGF + VEGF treatment groups suggesting that extracellular calcium influx is sufficient for CaMKII activation. CONCLUSION: VEGF inhibits PDGF-stimulated total calcium influx and, in particular, PDGF-stimulated intracellular calcium release in VSMC. The equivalent phosphorylation of PLCgamma1, PKC, and Akt suggests that the inhibitory mechanism by VEGF on calcium influx occurs downstream of these proximal mediators. The inhibition of intracellular calcium release did not inhibit CaMKII activity. VEGF may play an important role in modulating PDGF induced VSMC proliferation by specifically inhibiting intracellular calcium release in response to PDGF.

Arterial Occlusive Diseases↗

PubMed2005

The two-stage brachial artery-brachial vein autogenous fistula for hemodialysis: an alternative autogenous option for hemodialysis access.

The optimal dialysis access for the patient with chronic renal failure is considered to be an autogenous fistula; this is reflected in the recommendations of the National Kidney Foundation-Disease Outcomes Quality Initiatives (NKF-DOQI). If adequate superficial veins at the wrist or the forearm are not available, the next option is usually a prosthetic arteriovenous graft. In this case series, we describe our experience with an autogenous fistula constructed using the brachial vein. There were 20 patients over a 14-month period who were operated on for dialysis access. In these patients, no adequate superficial veins were found at operation. Instead of using a prosthetic graft, we performed a brachial artery-brachial vein fistula in two stages. The first stage involved a forearm anastomosis and then subsequently, weeks later, this fistula was "superficialized." Twenty patients underwent a brachial artery-brachial vein fistula. Of these patients, all had successful maturation of their fistula and after a minimum waiting period of 12 weeks for maturation; all but one were able to be successfully dialyzed through their fistula. One patient developed arm swelling due to previously placed subclavian vein pacemaker wires. None of the other patients developed arm swelling or vascular steal. The brachial artery-brachial vein fistula is a feasible option for hemodialysis access and we suggest that this option be considered before a prosthetic arteriovenous graft is inserted. Arm swelling and steal have not been a problem, and all patients have been able to have full dialysis through the fistula after appropriate maturation times.

Adult↗

PubMed2005

Vascular endothelial growth factor stimulates a novel calcium-signaling pathway in vascular smooth muscle cells.

BACKGROUND: Vascular endothelial growth factor (VEGF) is a potent vascular mitogen that selectively stimulates vascular smooth muscle cell (VSMC) migration through an unknown mechanism while having no effect on VSMC proliferation. It is known that VSMC migration and proliferation are dependent on the second messenger Ca2+ and, in particular, mitogen-stimulated Ca2+ influx. We hypothesized that the selective effect of VEGF on VSMC migration versus proliferation was a result of differential VEGF-stimulated Ca2+ signaling pathways. METHODS: Primary cultured human aortic smooth muscle cells (VSMCs) were grown to subconfluency and assigned to the following experimental groups: no stimulation, stimulation with platelet-derived growth factor-BB (PDGF-BB) (20 ng/mL) as positive control, and stimulation with VEGF165 (40 ng/mL). Total increase in [Ca2+]cyt and intracellular calcium release was quantified with the use of a fura-2 fluorescence assay. Assays for the following receptors VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1) and PDGFR-beta were performed by immunoprecipitation, while PLCgamma1, Akt 1/2, and phospholamban B phosphorylation were assessed with Western immunoblotting. RESULTS: VSMCs stimulated with VEGF165 exhibited no intracellular Ca2+ release, compared with a 75 +/- 30 nmol/L intracellular calcium release after PDGF-BB stimulation, (P < .02) VEGF165-stimulated VSMCs in Ca2+-containing media exhibited 192 +/- 26 nmol/L increase in [Ca2+]cyt, compared with 354 +/- 54 nmol/L increase after PDGF-BB stimulation (P < .02). VEGF165 did not phosphorylate PLCgamma1 after 1, 5, or 10 minutes of treatment. VEGF165 treatment did not result in PI3-K/Akt activation at 1-, 5-, or 10-minute time points. Calmodulin-dependent kinase II (CaMKII) was activated by both VEGF165 and PDGF-BB after 1 and 5 minutes of stimulation. The presence of the receptors VEGFR-1, VEGFR-2, and PDGFR-beta was confirmed in all experimental groups. CONCLUSIONS: VEGF induces extracellular calcium influx but no intracellular calcium release in VSMCs. This lack of intracellular Ca2+ release stems from the inability of VEGF165 to activate the PLCgamma1 cascade and IP3 receptor-mediated Ca2+ release. The lack of PI3-K/Akt activation at these time points indicates a novel extracellular Ca2+ influx pathway sufficient to activate CaMKII. A paradigm relating extracellular Ca2+ influx to CaMKII activation and migration is suggested and may account for the selective effects of VEGF on VSMC migration.

Aorta↗

PubMedDate not supplied

Occluded infrainguinal bypass graft: potential source of limb-threatening emboli.

Surgical bypass represents one of the chief treatment modalities for peripheral arterial occlusive disease. Despite improving techniques, graft occlusion accounts for the majority of these bypass failures. Once occluded, however, these grafts are thought to rarely pose a threat for future ischemic events. This report describes two patients with previously thrombosed grafts who subsequently presented with limb-threatening ischemia owing to peripheral embolization from the graft. Two patients with occluded grafts presented with ipsilateral limb-threatening acute ischemia. Both of these patients developed severe acute limb-threatening ischemia weeks to months after known graft thrombosis. Arteriography revealed peripheral embolization in each case. Both patients were operated on for disconnection of the thrombosed graft from the native circulation and have been free of recurrent symptoms. The occluded graft, although generally innocuous, can be a source of peripheral emboli, resulting in peripheral embolization and acute limb ischemia. Both patients in this report developed limb-threatening ischemia owing to embolization from the cul-de-sac of occluded prosthetic grafts. Due to the rarity of the condition and its associated morbidity and mortality, awareness and recognition of this phenomenon are critical. Operative disconnection is recommended if the embolism occurs downstream of the graft and no other embolic source can be identified.

Acute Disease↗