Variations in Structural Protein Expression and Endothelial Cell Proliferation in Relation to Clinical Manifestations of Cerebral Cavernous Malformations

Myristoylated alanine-rich C kinase substrate (MARCKS), as a specific protein kinase C (PKC) substrate, mediates PKC signaling through its phosphorylation and subsequent modification of its association with filamentous actin (F-actin) and calmodulin (CaM). PKC has long been implicated in cell proliferation, and recent studies have suggested that MARCKS may function as a cell growth suppressor. Therefore, in the present study, we investigated MARCKS protein expression, distribution, and phosphorylation in preconfluent and confluent bovine pulmonary microvascular endothelial cells (BPMEC) in the presence or absence of the vascular endothelial growth factor (VEGF). In addition, we examined functional alterations of MARCKS in these cells by studying the association of MARCKS with F-actin and CaM-dependent myosin light chain (MLC) phosphorylation. Our results indicate that MARCKS protein is downregulated during BPMEC proliferation. Decreased MARCKS association with F-actin, increased actin polymerization, and CaM-dependent MLC phosphorylation appear to mediate cell shape changes and motility during BPMEC growth. In contrast, VEGF stimulated MARCKS phosphorylation without alteration of protein expression during BPMEC proliferation, which may result in reduced interaction between MARCKS and actin or CaM, leading to actin reorganization and MLC phosphorylation. Our data suggest a regulatory role of MARCKS during endothelial cell proliferation.

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Inhibition of adenosine kinase induces expression of VEGF mRNA and protein in myocardial myoblasts

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2000.279.5.h2116 ◽

2000 ◽

Vol 279 (5) ◽

pp. H2116-H2123 ◽

Cited By ~ 17

Author(s):

Jian-Wei Gu ◽

Bruce R. Ito ◽

Amanda Sartin ◽

Nan Frascogna ◽

Michael Moore ◽

...

Keyword(s):

Cell Proliferation ◽

Endothelial Cell ◽

Protein Expression ◽

Kinase Inhibitor ◽

Umbilical Vein ◽

Adenosine Kinase ◽

Endothelial Cell Proliferation ◽

Cell Protein ◽

Vegf Mrna ◽

Vegf Protein

We tested whether increased endogenous adenosine produced by the adenosine kinase inhibitor GP-515 (Metabasis Therapeutics) can induce vascular endothelial growth factor (VEGF) expression in cultured rat myocardial myoblasts (RMMs). RMMs were cultured for 18 h in the absence (control) and presence of GP-515, adenosine (Ado), adenosine deaminase (ADA), or GP-515 + ADA. GP-515 (0.2–200 μM) caused a dose-related increase in VEGF protein expression (1.99–2.84 ng/mg total cell protein); control VEGF was 1.84 ± 0.05 ng/mg. GP-515 at 2 and 20 μM also increased VEGF mRNA by 1.67- and 1.82-fold, respectively. ADA (10 U/ml) decreased baseline VEGF protein levels by 60% and completely blocked GP-515 induction of VEGF. Ado (20 μM) and GP-515 (20 μM) caused a 59 and 39% increase in VEGF protein expression and a 98 and 33% increase in human umbilical vein endothelial cell proliferation, respectively, after 24 h of exposure. GP-515 (20 μM) had no effect on VEGF protein expression during severe hypoxia (1% O2) but increased VEGF by an additional 27% during mild hypoxia (10% O2). These results indicate that raising endogenous levels of Ado through inhibition of adenosine kinase can increase the expression of VEGF and stimulate endothelial cell proliferation during normoxic and hypoxic conditions.

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Modulation of VEGF-induced endothelial cell cycle protein expression through cyclic AMP hydrolysis by PDE2 and PDE4

Thrombosis and Haemostasis ◽

10.1160/th03-12-0768 ◽

2004 ◽

Vol 92 (09) ◽

pp. 634-645 ◽

Cited By ~ 33

Author(s):

Laure Favot ◽

Thérèse Keravis ◽

Claire Lugnier

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Endothelial Cell ◽

Protein Expression ◽

Map Kinase ◽

Cyclin D1 ◽

Cyclin A ◽

Pde4 Inhibitor ◽

Endothelial Cell Proliferation ◽

Cell Cycle Protein

SummaryEndothelial cell proliferation in response to VEGF plays an important role in physiological and pathological angiogenesis. The role of PDE2 and PDE4 in VEGF-induced proliferation in HUVEC was investigated: 1) VEGF increased cAMP-hydrolytic activity by up-regulating the expression of PDE2 and PDE4 isozymes; 2) VEGF increased progression in cell cycle with an increase in p42/p44 MAP kinase, cyclin A and cyclin D1 expressions and with a decrease in p21waf1/cip1 and p27kip1 expressions; 3) EHNA (20 µM), a selective PDE2 inhibitor, RP73401 (10 µM), a selective PDE4 inhibitor blocked the VEGF-induced increase in p42/p44 MAP kinase expression; 4) RP73401, but not EHNA, blocked the VEGF-induced increase in cyclin A and decrease in p27kip1 expressions; 5) EHNA, contrary to RP73401, enhanced the VEGF-induced increase of cyclin A and decrease of p27kip1. EHNA and RP73401 together blocked the VEGF-induced increase in cyclin D1 and decrease in p21waf1/cip1 expressions; Inhibition of VEGF-upregulated PDE2 and PDE4 reversed the VEGF-induced alterations in cell cycle protein expression, bringing back endothelial cells to a non-proliferating status. Consequently, PDE2 and PDE4 inhibitions were able to inhibit VEGF-induced endothelial cell proliferation by restoring cell cycle key protein expression, and might thus be useful in excessive angiogenesis. Furthermore, the differences between PDE2 and PDE4 effects may suggest compartmentalized effects.

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