Intracellular Ca ²⁺ Handling in Vascular Smooth Muscle Cells Is Affected by Proliferation

Abstract

Abstract-Despite intensive interest in the dedifferentiation process of vascular smooth muscle cells, very little data are available on intracellular Ca 2ϩ signaling. The present study was designed to investigate the evolution of the intracellular Ca 2ϩ pools when rat aortic smooth muscle cells (RASMCs) proliferate and to define the mechanisms involved in the functional alterations. RASMCs were cultured in different conditions, and [Ca 2ϩ ] i was measured by use of fura 2. Expression of the sarco(endo)plasmic reticulum Ca 2ϩ pumps (SERCA2a and SERCA2b), Ca 2ϩ channels, the ryanodine receptor (RyR), and the inositol trisphosphate receptor (IP3R) was studied by reverse transcription-polymerase chain reaction and immunofluorescence. Antibodies specific for myosin heavy chain isoforms were used as indicators of the differentiation state of the cell, whereas an anti-proliferating cell nuclear antigen antibody was a marker of proliferation. SERCA2a, SERCA2b, RyR3, and IP3R-1 mainly were present in the aorta in situ and in freshly isolated RASMCs. These cells used the 2 types of Ca 2ϩ channels to release Ca 2ϩ from a common thapsigargin-sensitive store. Proliferation of RASMCs, induced by serum or by platelet-derived growth factor-BB, resulted in the disappearance of RyR and SERCA2a mRNAs and proteins and in the loss of the caffeine-and ryanodine-sensitive pool. The differentiated nonproliferative phenotype was maintained in low serum or in cells cultured at high density. In these conditions, RyR and SERCA2a were also present in RASMCs. Thus, expression of RyR and SERCA2a is repressed by cell proliferation, inducing loss of the corresponding Ca 2ϩ pool. In arterial smooth muscle, Ca 2ϩ release through RyRs is involved in vasodilation, and suppression of the ryanodine-sensitive pool might thus alter the control of vascular tone.

Endothelin-Induced Sarcoplasmic Reticulum Calcium Depletion Waves in Vascular Smooth Muscle Cells

“…The fact that the depletion wave is blocked by inhibition of IP 3 R indicates that like other vascular smooth muscle Ca 2+ waves, the underlying mechanism is indeed mediated by CICR at IP 3 R [10,17,19]. This notion was further corroborated by the observation that our cultured rat SMCs failed to respond to caffeine treatment, confirming the lack of functional RyRs in these cells (data not shown), which is in agreement with previous report by Vallot et al [20].…”

Sarco/Endoplasmic Reticulum Ca 2+ -ATPase Gene Transfer Reduces Vascular Smooth Muscle Cell Proliferation and Neointima Formation in the Rat

“…First, SERCA2a expression is markedly downregulated in highly proliferating VSMCs in vivo in a model of balloon injury of the rat carotid artery. Second, SERCA2a is repressed in vitro in seruminduced proliferation 9 and in proliferation induced by PDGF 9 or very low density lipoproteins. 7 Finally, we show here that overexpression of SERCA2a inhibits mitogenic stimuli mediated-proliferation of VSMC and blocks balloon injuryinduced neointimal VSMC proliferation in vivo.…”

“…2ϩ concentration in proliferating VSMC are supported by long-term alterations in the levels of Ca 2ϩ -handling proteins such as loss of RyR and SERCA2a, 9 replacement of L-type voltage-operated Ca 2ϩ channels by T-type voltage-operated Ca 2ϩ channels, loss of the plasma membrane Ca 2ϩ pumps, and upregulation of the transient receptor potential Ca 2ϩ channels. 4 Any chronic alterations in the spatio-temporal pattern of Ca 2ϩ signals should alter gene expression by activating different kinases and phosphatases, modulating Ca 2ϩ -regulated transcription factors such as NFAT (nuclear factor of activated T lymphocytes).…”

“…6,7,14 -16 Several previous studies have suggested that SERCA2a is involved in the control of proliferation and growth: transgenic mice with only one allele of the ATP2a2 gene (SERCA2) develop numerous cancers of the upper digestive tract and skin, and cardiac hypertrophy 17,18 ; low levels of SERCA2a are associated with cardiac hypertrophy both in humans and animals 19 -21 ; mice in which SERCA2a has been replaced by SERCA2b develop cardiac hypertrophy 22 ; finally, loss of SERCA2a is associated with VSMC proliferation. 7,9 Similarly, only SERCA2b is present in proliferating BC3H1 muscle cells whereas SERCA1, SERCA2a, and SERCA2b are present in quiescent differentiated BC3H1 muscle cells. 23 We hypothesized that increasing the rate of SR Ca 2ϩ uptake by restoring SERCA2a expression might inhibit VSMC proliferation and prevent neointima formation induced by injury.…”

“…Both SERCA2a and SERCA2b were expressed in these cells, as described. 9 Proliferating cells did not contain SERCA2a and only low levels of SM1 and SM2, but still expressed NM-B and smooth muscle cell specific markers such as calponin and caldesmon ( Figure 1S). Thus, at the time of infection, VSMCs were in the synthetic phenotype.…”