Chronic antagonism of the mineralocorticoid receptor ameliorates hypertension and end organ damage in a rodent model of salt-sensitive hypertension.

We investigated the effects of chronic mineralocorticoid receptor blockade with eplerenone on the development and progression of hypertension and end organ damage in Dahl salt-sensitive rats. Eplerenone significantly attenuated the progressive rise in systolic blood pressure (SBP) (204 ± 3 vs. 179±3 mmHg, p < 0.05), reduced proteinuria (605.5 ± 29.6 vs. 479.7 ± 26.1 mg/24h, p < 0.05), improved injury scores of glomeruli, tubules, renal interstitium, and vasculature in Dahl salt-sensitive rats fed a high-salt diet. These results demonstrate that mineralocorticoid receptor antagonism provides target organ protection and attenuates the development of elevated blood pressure (BP) in a model of salt-sensitive hypertension.

Disruption of protein arginine N-methyltransferase 2 regulates leptin signaling and produces leanness in vivo through loss of STAT3 methylation.

RATIONALE: Arginine methylation by protein N-arginine methyltransferases (PRMTs) is an important posttranslational modification in the regulation of protein signaling. PRMT2 contains a highly conserved catalytic Ado-Met binding domain, but the enzymatic function of PRMT2 with respect to methylation is unknown. The JAK-STAT pathway is proposed to be regulated through direct arginine methylation of STAT transcription factors, and STAT3 signaling is known to be required for leptin regulation of energy balance. OBJECTIVE: To identify the potential role of STAT3 arginine methylation by PRMT2 in the regulation of leptin signaling and energy homeostasis. METHODS AND RESULTS: We identified that PRMT2(-/-) mice are hypophagic, lean, and have significantly reduced serum leptin levels. This lean phenotype is accompanied by resistance to food-dependent obesity and an increased sensitivity to exogenous leptin administration. PRMT2 colocalizes with STAT3 in hypothalamic nuclei, where it binds and methylates STAT3 through its Ado-Met binding domain. In vitro studies further clarified that the Ado-Met binding domain of PRMT2 induces STAT3 methylation at the Arg31 residue. Absence of PRMT2 results in decreased methylation and prolonged tyrosine phosphorylation of hypothalamic STAT3, which was associated with increased expression of hypothalamic proopiomelanocortin following leptin stimulation. CONCLUSIONS: These data elucidate a molecular pathway that directly links arginine methylation of STAT3 by PRMT2 to the regulation of leptin signaling, suggesting a potential role for PRMT2 antagonism in the treatment of obesity and obesity-related syndromes.

KIS protects against adverse vascular remodeling by opposing stathmin-mediated VSMC migration in mice.

Vascular proliferative diseases are characterized by VSMC proliferation and migration. Kinase interacting with stathmin (KIS) targets 2 key regulators of cell proliferation and migration, the cyclin-dependent kinase inhibitor p27Kip1 and the microtubule-destabilizing protein stathmin. Phosphorylation of p27Kip1 by KIS leads to cell-cycle progression, whereas the target sequence and the physiological relevance of KIS-mediated stathmin phosphorylation in VSMCs are unknown. Here we demonstrated that vascular wound repair in KIS-/- mice resulted in accelerated formation of neointima, which is composed predominantly of VSMCs. Deletion of KIS increased VSMC migratory activity and cytoplasmic tubulin destabilizing activity, but abolished VSMC proliferation through the delayed nuclear export and degradation of p27Kip1. This promigratory phenotype resulted from increased stathmin protein levels, caused by a lack of KIS-mediated stathmin phosphorylation at serine 38 and diminished stathmin protein degradation. Downregulation of stathmin in KIS-/- VSMCs fully restored the phenotype, and stathmin-deficient mice demonstrated reduced lesion formation in response to vascular injury. These data suggest that KIS protects against excessive neointima formation by opposing stathmin-mediated VSMC migration and that VSMC migration represents a major mechanism of vascular wound repair, constituting a relevant target and mechanism for therapeutic interventions.

GA-binding protein regulates KIS gene expression, cell migration, and cell cycle progression.

The cyclin-dependent kinase inhibitor p27(Kip1) arrests cell cycle progression through G1/S phases and is regulated by phosphorylation of serine/threonine residues. Recently, we identified the serine/threonine kinase, KIS, which phosphorylates p27(Kip1) on serine 10 leading to nuclear export of p27(Kip1) and protein degradation. However, the molecular mechanisms of transcriptional activation of the human KIS gene and its biological activity are not known. We mapped the transcription initiation site approximately 116 bp 5' to the translation start site, and sequences extending to -141 were sufficient for maximal promoter activity. Mutation in either of two Ets-binding sites in this region resulted in an approximately 75-80% decrease in promoter activity. These sites form at least 3 specific complexes, which contained GA-binding protein (GABP). Knocking down GABPalpha by siRNA in vascular smooth muscle cells (VSMCs) diminished KIS gene expression and reduced cell migration. Correspondingly, in serum stimulated GABPalpha-deficient mouse embryonic fibroblasts (MEFs), KIS gene expression was also significantly reduced, which was associated with an increase in p27(Kip1) protein levels and a decreased percentage of cells in S-phase. Consistent with these findings, following vascular injury in vivo, GABPalpha-heterozygous mice demonstrated reduced KIS gene expression within arterial lesions and these lesions were significantly smaller compared to GABP+/+ mice. In summary, serum-responsive GABP binding to Ets-binding sites activates the KIS promoter, leading to KIS gene expression, cell migration, and cell cycle progression.

The arginine methyltransferase PRMT2 binds RB and regulates E2F function.

The retinoblastoma gene product (RB) is an important regulator of E2F activity. RB recruits a number of proteins, including HDACs, SWI/SNF complex, lysine methyl transferase (SUV39H1) and DNA methyltransferase (DNMT1), all of which negatively regulate E2F activity with RB. Here, we show that RB interacts with PRMT2, a member of the protein arginine methyltransferase family, to regulate E2F activity. PRMT2 directly bound and interacted with RB through its AdoMet binding domain, in contrast to other PRMT proteins, including PRMT1, PRMT3 and PRMT4. In reporter assays, PRMT2 repressed E2F1 transcriptional activity in an RB-dependent manner. PRMT2 formed a ternary complex with E2F1 in the presence of RB. To further explore the role of endogenous PRMT2 in the regulation of E2F activity, the PRMT2 gene was ablated in mice by gene targeting. Compared with PRMT2(+/+) mouse embryonic fibroblasts (MEFs), PRMT2(-/-) MEFs demonstrated increased E2F activity and early S phase entry following release of serum starvation. Vascular injury to PRMT2(-/-) arteries results in a hyperplastic response, consistent with increased G1-S phase progression. Taken together, these findings demonstrate a novel mechanism for the regulation of E2F activity by a member of the protein arginine methyltransferase family.

Bone marrow-derived immune cells regulate vascular disease through a p27(Kip1)-dependent mechanism.

The cyclin-dependent kinase inhibitors are key regulators of cell cycle progression. Although implicated in carcinogenesis, they inhibit the proliferation of a variety of normal cell types, and their role in diverse human diseases is not fully understood. Here, we report that p27(Kip1) plays a major role in cardiovascular disease through its effects on the proliferation of bone marrow-derived (BM-derived) immune cells that migrate into vascular lesions. Lesion formation after mechanical arterial injury was markedly increased in mice with homozygous deletion of p27(Kip1), characterized by prominent vascular infiltration by immune and inflammatory cells. Vascular occlusion was substantially increased when BM-derived cells from p27(-/-) mice repopulated vascular lesions induced by mechanical injury in p27(+/+) recipients, in contrast to p27(+/+) BM donors. To determine the contribution of immune cells to vascular injury, transplantation was performed with BM derived from RAG(-/-) and RAG(+/+) mice. RAG(+/+) BM markedly exacerbated vascular proliferative lesions compared with what was found in RAG(-/-) donors. Taken together, these findings suggest that vascular repair and regeneration is regulated by the proliferation of BM-derived hematopoietic and nonhematopoietic cells through a p27(Kip1)-dependent mechanism and that immune cells largely mediate these effects.

Gene transfer strategies to inhibit neointima formation.

Vascular smooth muscle cell (VSMC) proliferation after arterial injury results in neointima formation and plays an important role in the pathogenesis of restenosis after angioplasty, in-stent restenosis, vascular bypass graft occlusion, and allograft vasculopathy. Major progress has been made recently in elucidating the cellular and molecular mechanisms underlying neointima formation. However, no known curative treatment currently exists. In cases in which pharmacologic and surgical interventions have had limited success, gene therapy remains a potential strategy for the treatment of such vascular proliferative diseases. To date, recombinant adenoviral vectors continue to be the most efficient methods of gene transfer into the arterial wall. However, concerns over the safety of using viral vectors in a clinical situation have inspired the considerable progress that has been made in improving both viral and nonviral modes of gene transfer. This review discusses some of the recent insights and outstanding progress in vascular gene therapeutic approaches to inhibit neointima both from a biologic and therapeutic perspective.

A growth factor-dependent nuclear kinase phosphorylates p27(Kip1) and regulates cell cycle progression.

The cyclin-dependent kinase inhibitor, p27(Kip1), which regulates cell cycle progression, is controlled by its subcellular localization and subsequent degradation. p27(Kip1) is phosphorylated on serine 10 (S10) and threonine 187 (T187). Although the role of T187 and its phosphorylation by Cdks is well-known, the kinase that phosphorylates S10 and its effect on cell proliferation has not been defined. Here, we identify the kinase responsible for S10 phosphorylation as human kinase interacting stathmin (hKIS) and show that it regulates cell cycle progression. hKIS is a nuclear protein that binds the C-terminal domain of p27(Kip1) and phosphorylates it on S10 in vitro and in vivo, promoting its nuclear export to the cytoplasm. hKIS is activated by mitogens during G(0)/G(1), and expression of hKIS overcomes growth arrest induced by p27(Kip1). Depletion of KIS using small interfering RNA (siRNA) inhibits S10 phosphorylation and enhances growth arrest. p27(-/-) cells treated with KIS siRNA grow and progress to S/G(2 )similar to control treated cells, implicating p27(Kip1) as the critical target for KIS. Through phosphorylation of p27(Kip1) on S10, hKIS regulates cell cycle progression in response to mitogens.

Both ICAM-1- and VCAM-1-integrin interactions are important in mediating monocyte adhesion to human saphenous vein.

Atherosclerosis underlies late occlusion of human saphenous vein (HSV) coronary artery bypass grafts. Monocyte infiltration is implicated, but its mechanisms are unclear given that HSV normally expresses the ICAM-1 but not the VCAM-1 adhesion molecule. To define the mechanisms underlying monocyte adhesion, samples of HSV taken from coronary artery bypass graft patients were co-cultured with human monocytes and adherent monocytes were quantified by immunocytochemistry for CD68 on transverse sections. Pre-treatment of veins with anti-ICAM-1 antibodies reduced monocyte adhesion (monocytes/mm of section) from 7.2 +/- 1.5 to 3.1 +/- 0.7 (p < 0.05, n = 6), but the effect of anti-VCAM-1 was not significant (4.1 +/- 0.7). Paradoxically, pre-treatment of monocytes with either anti-beta(2)-integrins, the counter-receptor of ICAM-1, or anti-alpha(4) integrins, the counter-receptor of VCAM-1, significantly reduced adhesion (1.8 +/- 0.6 and 2.4 +/- 0.7, respectively, p < 0.05). These results were clarified by immunocytochemistry, which confirmed that VCAM-1 expression was absent in harvested vein but was induced in the endothelium during co-culture. Consistent with this, when anti-ICAM-1 or anti-VCAM-1 was present throughout co-culture, either of them reduced adhesion (from 4.2 +/- 0.9 to 2.3 +/- 0.5 and 2.2 +/- 0.4, respectively, p < 0.02, n = 8) and there was no further effect of adding both (2.0 +/- 0.5). These results demonstrate that both ICAM-1/beta(2) and VCAM-1/alpha(4) integrin interactions mediate monocyte adhesion to HSV, possibly as part of a common pathway. These experiments imply that either integrin might be targeted to reduce monocyte infiltration into HSV grafts.

Role of nuclear factor-kappa B activation in metalloproteinase-1, -3, and -9 secretion by human macrophages in vitro and rabbit foam cells produced in vivo.

Metalloproteinase secretion by macrophages is believed to play a key role in the matrix degradation that underlies atherosclerotic plaque instability and aneurysm formation. We studied the hypothesis that nuclear factor-kappaB (NF-kappaB), a transcription factor, is necessary for metalloproteinase secretion and, hence, is a target for pharmacological intervention. Adenovirus-mediated gene transfer of the inhibitory NF-kappaB subunit, I-kappa Balpha, was achieved into human monocyte-derived macrophages in vitro and into foam cells produced in vivo in cholesterol-fed rabbits. Human macrophages and rabbit foam cells secreted matrix-degrading metalloproteinase (MMP)-9 without further stimulation, and this was not inhibited by I-kappaBalpha (11+/-16% and 8+/-10%, respectively; P> 0.05). MMP-1 secretion from human macrophages increased in response to recombinant human CD40 ligand and was inhibited 92+/-5% by I-kappaBalpha (n=3, P<0.05). Rabbit foam cells secreted MMP-1 and -3 without further stimulation, and this was inhibited 83+/-12% and 69+/-11%, respectively, by I-kappaBalpha (n=6 or 7, P<0.001). I-kappaBalpha did not significantly affect the expression or activity of tissue inhibitor of metalloproteinases-1 or -2. Overexpression of I-kappaBalpha inhibited collagenolytic and beta-caseinolytic activity by 42+/-2% and 41+/-7%, respectively (n=3, P<0.05). Secretion of MMP-1 and MMP-3 from macrophages stimulated in vitro or in vivo depends on the activation of NF-kappaB. Because the inhibition of NF-kappaB reduces proteolytic activity, it appears to be an attractive pharmacological target in unstable atheromas.