Heparin Inhibits Phosphorylation and Autonomous Activity of Ca2+/Calmodulin-Dependent Protein Kinase II in Vascular Smooth Muscle Cells

Abstract

Vascular smooth muscle cell (VSMC) hyperproliferation is a characteristic feature of both atherosclerosis and restenosis seen after vascular surgery. A number of studies have shown that heparin inhibits VSMC proliferation in vivo and in culture. To test our hypothesis that heparin mediates its antiproliferative effect by altering Ca 2؉ regulated pathways involved in mitogenic signaling in VSMC, we analyzed the effect of heparin on multifunctional Ca 2؉ /calmodulin dependent protein kinase II (CaM kinase II) which is abundantly expressed in VSMC. Using activity assays, radioactive labeling, and immunoprecipitation it was found that heparin inhibits the overall phosphorylation of the ␦-subunit of CaM kinase II which is consistent with inhibition of autophosphorylation-dependent, Ca 2؉ /calmodulin-independent CaM kinase II activity. This effect was less evident in heparinresistant cells, consistent with a role for CaM kinase II in mediating the antiproliferative effect of heparin. Diseases of the heart and blood vessels are the leading cause of death in the United States and are responsible for almost half the deaths recorded each year. Most of these are due to atherosclerosis and its ensuing complications, which include hypertension, myocardial infarction, and gangrene. Hyperplasia of vascular smooth muscle cells (VSMC) is the hallmark of early atherogenesis and is considered the major cause of the high failure rate of vascular surgical procedures such as angioplasty, coronary artery bypass grafts, and heart transplants.1 The severity and prevalence of the problems associated with VSMC hyperplasia have provided the impetus to develop and characterize inhibitors of VSMC proliferation.Heparin and heparan sulfates are one such class of VSMC proliferation inhibitors.2 Work in our laboratory and by others has supported the antiproliferative role of heparin in animals and in culture systems.3 Heparin suppresses VSMC and mesangial cell proliferation while most other cells are unaffected. 4 Several studies point to the possibility that heparin blocks VSMC and mesangial cell proliferation via alterations in mitogenic signal transduction pathways.5-7 Heparin binds to specific, saturable high-affinity binding sites on VSMC and is internalized by receptor-mediated endocytosis. 8 Heparin has also been shown to selectively block the PKC pathway of mitogenic signaling as well as the phosphorylation and activation of MAPK.5,9 This is followed by a rapid down-regulation of mRNA levels of genes involved in growth regulation (eg, c-fos, c-jun, myb, and myc), thus blocking cells in the G 1 phase of the cell cycle.10 Despite these studies a causeeffect relationship has not yet been established.An important aspect of cellular signaling involves Ca 2ϩ mobilization in response to various stimuli. Calcium regulates a number of important functions in cells including proliferation, migration, contraction, and transcription, and the ability of heparin to block calcium transients in SMC has been appreciated for more than a decade. Several stu...

Effects of heparin on the production of homocysteine-induced extracellular matrix metalloproteinase-2 in cultured rat vascular smooth muscle cells

“…Mori et al (34) suggested that histidine-rich glycoprotein, in combination with the zinc ion, plays a role in modulating the SMC growth response in pathophysiological states and explains the lack of success of heparin as a therapeutic antirestenosis drug in clinical trials. Mishra-Gorur et al (35) showed that alterations in calcium-mediated mitogenic signalling pathways may be involved in the antiproliferative mechanism of action of heparin. He et al (36) showed that a heparinoid at 1.6 mg/mL to 0.05 mg/mL significantly inhibits VSMC proliferation induced by FCS (10%), basic fibroblast growth factor or interleukin-1.…”

miR-143 and miR-145

“…miR-145 represses Klf4, which otherwise interacts with SRF and also represses myocardin, 40 and calmodulin kinase II-␦ (CamkII-␦), which was shown to be involved in multiple events including neointimal proliferation. 41,42 miR-143 represses Elk-1 (Ets LiKe gene 1), which competes with myocardin to bind SRF and exhibits an inhibitory effect on smooth muscle differentiation. 43 Klf4 plays a key role in regulating VSMC phenotype because it antagonizes proliferation, facilitates migration, and downregulates VSMC differentiation marker genes.…”

Regulation of Vascular Smooth Muscle Proliferation by Heparin

“…MAPK cascade has also shown to be required for p27 kip1 down-regulation and S phase entry in fibroblasts and epithelial cells (80). Inhibitory effects of heparin on activation of calmodulin-dependent protein kinase II have been documented by Mishra-Gorur et al (81,82) in VSMC. Recently, it has been shown that inhibition of calmodulindependent protein kinase II in NIH 3T3 cells enhances association of p27 kip1 with Cdk2 and thus causes a G 1 arrest (83).…”