Proceedings of The Physiological Society

Physiology 2014 (London, UK) (2014) Proc Physiol Soc 31, PCB191

Poster Communications

Human mesenchimal stem cells transplant failed to restore vasodilator potential in vascular tissues from diabetic rats but succeeded in spontaneously hypertensive rats

A. I. Soloviev1, I. Ivanova2, T. Novokhatska3, K. Klymenko4

1. Experimental Therapeutics, Institute of Pharmacology&Toxicology, Kiev, Ukraine. 2. Experimental Therapeutics, Institute of Pharmacology&Toxicology, Kiev, Ukraine. 3. Experimental Therapeutics, Institute of Pharmacology&Toxicology, Kiev, Ukraine. 4. Experimental Therapeutics, Institute of Pharmacology&Toxicology, Kiev, Ukraine.

Potassium conductance and endothelium-dependent vascular relaxation are known to be altered in both spontaneously hypertensive (SHR) and diabetic rats. Additionally, an overproduction of reactive oxygen species (ROS) and a high level of protein kinase C (PKC) activity are common features for arterial hypertension and diabetes. The main goal of this study was to evaluate the therapeutic potential of human mesenchimal stem cells (hMSCs) to restore vasodilator potential in SHR and diabetic rats. The rats were anesthetized with ketamine (45 mg/kg b.w., i.p.) and xylazine (5 mg/kg b.w., i.p.). Experimental design of the study comprised patch-clamp technique, standard dilation ACh-test and ROS production measurement in vascular tissues. Systolic arterial blood pressure was measured innon-anesthetized rats using tail cuff sphygmomanometer S-2 (Hugo-Sachs Electronic, Germany). Bone marrow was aspirated in heparin from the sternum of healthy volunteers after informed consent. To reduce the burning effect of lidocaine solution, local anesthesia was achieved by use of 1% lidocaine (up to 4 µg/kg) in 1% sodium bicarbonate solution injected subcutaneously and into the periosteum. hMSCs were separated using negative selection procedure with monoclonal antibodies. The isolated hMSCs were centrifugated, then resuspended and cultivated 20 - 32 days in MesenCult medium with recombinant human growth factors and appropriate Mesenchymal Stem Cell Stimulatory Supplements. Then hMSCs were transplanted intravenously to the rats in a single dose of 16-20x106 cells per rat. Phenotypic analysis confirmed that hMSCs used for transplantation were free of hematopoetic contamination and strongly positive for the MSCs specific surface markers. hMSCs transplantation had no effect on glucose plasma level and systolic blood pressure in both diabetic rats and SHR. Maximal Ach-induced relaxation (Rmax) in SHR thoracic aorta smooth muscle (SM) and their sensitivity to Ach were decreased (Rmax=32,2±3,4% and pD2 (-log EC50)=6,4 ± 0,1, n=10, p<0,01 vs control). hMSCs treatment led to repair in Rmax and pD2 up to 60,5±4,6 % (n=10, p<0,01) and 7,8±0,2 (n=10, p<0,01), respectively. Rmax and pD2 in diabetic rats were 49,5± 4,6 % and 6,9±0,04 (n=10, p<0,01 vs control), respectively. hMSCs was without effect on Rmax but increased the sensitivity to Ach (pD2=7,2±0,05, n=10, p<0,01 vs control).Outward potassium current (IK) in SHR was decreased resulting in a lower current density (38,7±2,0 pA/pF, n=9, p<0,01 vs control) but impressively increased after hMSC treatment (IK= 110,1± 5,1 pA/pF, n=9, p<0,05 vs control). In contrast to SHR, diabetic vascular tissues appeared to be resistant to hMSCs - IK was without difference before and after hMSCs administration - 40,1± 3,6 pA/pF and 32,5±3,7 pA/pF(n=9, p >0,05), respectively. In conclusion, the loss of hMSCs effectiveness under diabetes as compared to SHR may be due to negative effect of hyperglycemia and ROS overproduction on hMSCs homing or/and paracrine effects. Vascular rings from diabetic rats showed an increased ROS production under diabetes in comparison to those from healthy animals and even in SHR. It is known that hMSCs obtained from bone marrow are extremely sensitive to ROS (Soloviev et al., 2012) and hyperglycemia (Dhanasekaran et al., 2013) which, in turn, leads to ROS overproduction.

Where applicable, experiments conform with Society ethical requirements