Electrophysiological and contractile evidence of the ability of human mesenchymal stromal cells to correct vascular malfunction in rats after ionizing irradiation.

The effect of intravenous administration of human mesenchymal stromal stem cells (hMSC) has been evaluated by means of large-conductance calcium-dependent potassium channel (BK(Ca)) activity measurements in thoracic aorta smooth muscle cells (SMC) obtained from non-fatal whole-body irradiated rats, using the patch clamp technique in whole-cell modification, and the standard acetylcholine (ACh) test to evaluate functional endothelium integrity using SM contractile recordings. Myofilament calcium sensitivity was estimated using simultaneous contractile recordings versus [Ca(2+)](i). Arterial blood was measured in intact and irradiated rats before and after hMSC administration. Stimulation of isolated SMC from the control group of animals with depolarizing voltage steps showed that outward K(+) currents sensitive to the BK(Ca) inhibitor paxilline were expressed. Outward currents in SMC obtained from irradiated animals were significantly reduced on the 30th day of post-irradiation. Irradiation led to a significant elevation in arterial blood pressure and reduced ACh-induced relaxation responses in irradiated rats as compared with the control group. Simultaneous measurements of contractile force and [Ca(2+)](i) showed that myofilament Ca(2+) sensitivity had increased following irradiation. Intravenously injected hMSC effectively restored BK(Ca) current and the amplitude of ACh-induced endothelium-dependent vasodilatation in vascular tissues obtained from post-irradiated rats. SMC obtained from irradiated rats treated with hMSC demonstrated a significantly increased paxilline-sensitive component of outward potassium currents, indicating that BK(Ca) activity had been restored. hMSC administration normalized increased blood pressure and myofilament Ca(2+) sensitivity in irradiated animals. When administered to healthy rats, hMSC were without effects on either of these. This study does not provide any immunohistochemical proof of hMSC engraftment in the host rats. PCR analysis showed that hMSCs were negative for hematopoietic cell markers and positive for hMSC markers. There were no clinical signs of graft-versus-host disease throughout the experimental period of 30 days. The data obtained suggest that hMSC demonstrate a clearly expressed ability to normalize vascular function damaged following irradiation, i.e. to reduce an elevated arterial blood pressure and myofilament Ca(2+) sensitivity, and to repair BK(Ca) function and endothelium-dependent relaxation in vascular tissues obtained from irradiated animals. Thus, hMSC seem to be worthwhile therapeutic approach in cases of ionizing irradiation accident or radiation beam therapy.

Functional and molecular consequences of ionizing irradiation on large conductance Ca2+-activated K+ channels in rat aortic smooth muscle cells.

AIMS: The goal of this study was to evaluate the influence of gamma-irradiation on Ca(2+)-activated K(+) channel (BK(Ca)) function and expression in rat thoracic aorta. MAIN METHODS: Aortic cells or tissues were studied by the measurement of force versus [Ca(2+)](i), patch-clamp technique, and RT-PCR. KEY FINDINGS: Stimulation of smooth muscle cells with depolarizing voltage steps showed expression of outward K(+) currents. Paxilline, an inhibitor of BK(Ca) channels, decreased outward K(+) current density. Outward currents in smooth muscle cells obtained from irradiated animals 9 and 30 days following radiation exposure demonstrated a significant decrease in K(+) current density. Paxilline decreased K(+) current in cells obtained 9 days, but was without effect 30 days after irradiation suggesting the absence of BK(Ca) channels. Aortic tissue from irradiated animals showed progressively enhanced contractile responses to phenylephrine in the post-irradiation period of 9 and 30 days. The concomitant Ca(2+) transients were significantly smaller, as compared to tissues from control animals, 9 days following irradiation but were increased above control levels 30 days following irradiation. Irradiation produced a decrease in BK(Ca) alpha- and beta(1)-subunit mRNA levels in aortic smooth muscle cells suggesting that the vasorelaxant effect of these channels may be diminished. SIGNIFICANCE: These results suggest that the enhanced contractility of vascular tissue from animals exposed to radiation may result from an increase in myofilament Ca(2+) sensitivity in the early post-irradiation period and a decrease in BK(Ca) channel expression in the late post-irradiation period.

Ionizing radiation alters myofilament calcium sensitivity in vascular smooth muscle: potential role of protein kinase C.

Radiation exposure increases vascular responsiveness, and this change involves endothelial damage, as well as direct effects on vascular smooth muscle. In this study, we tested the hypothesis that myofilament Ca(2+) sensitivity in vascular smooth muscle is increased from single whole body gamma irradiation (6 Gy). We measured contractile responses from intact and permeabilized rat thoracic aortic rings combined with cytosolic Ca(2+) ([Ca(2+)](i)) measurements. The sensitivity to KCl and phenylephrine increased significantly in tissues from animals on the 9th and 30th days postirradiation compared with control. Irradiation also significantly increased Ca(2+) sensitivity in beta-escin permeabilized smooth muscle on the 9th and 30th days postirradiation. Inhibitors of protein kinase C, chelerythrine, and staurosporine, had no effect on the pCa-tension curves in control permeabilized tissues but significantly decreased Ca(2+) sensitivity in permeabilized tissues on the 9th and 30th days postirradiation. Phorbol dibutyrate (PDBu, 10(-7) M) increased Ca(2+) sensitivity in control skinned smooth muscle but was without effect in irradiated vascular rings. Simultaneous measurement of contractile force and [Ca(2+)](i) showed that myofilament Ca(2+) sensitivity defined as the ratio of force change to [Ca(2+)](i) significantly increased following gamma-irradiation. PDBu (10(-6) M) stimulation of intact aorta produced a sustained contraction, while the increase in [Ca(2+)](i) was transient. In irradiated tissues, PDBu-induced contractions were greater than those seen in control tissues but there was no elevation in [Ca(2+)](i). Taken together, these data strongly support the hypothesis that irradiation increases the sensitivity of vascular smooth muscle myofilaments to Ca(2+) and this effect is dependent on activation of protein kinase C.