Antonio Díez-Juan

Overexpression of p27Kip1 by doxycycline-regulated adenoviral vectors inhibits endothelial cell proliferation and migration and impairs angiogenesis

Formation of new blood vessels in the adult animal (i.e., angiogenesis) is an important event for tissue repair and for tumor growth and metastasis. Angiogenesis involves the migration and proliferation of endothelial cells. We have investigated the role of the growth suppressor p27Kip1 (p27) on endothelial cell function in vitro and angiogenesis in vivo. We have generated Ad‐TetON, a replication‐deficient adenovi‐rus that constitutively expresses the reverse tet‐respon‐sive transcriptional activator, and Ad‐TRE‐p27, which drives expression of p27 under the control of the tet response element. Western blot analysis demonstrated doxycycline‐dependent overexpression of p27 in human umbilical vein endothelial cells (HUVECs) coinfected with Ad‐TetON and Ad‐TRE‐p27, which resulted in a marked inhibition of DNA replication and cell migration in vitro. Inducible overexpression of p27 in cultured HUVECs inhibited the formation of tubelike structures and, when applied in a murine model of hind limb ischemia, reduced hind limb blood flow recovery and capillary density. These findings thus underscore a novel role of p27 in regulating endothelial cell migration in vitro and angiogenesis in vivo, suggesting a novel anti‐angiogenic therapy based on inducible p27 overexpression.

The growth suppressor p27Kip1 protects against diet-induced atherosclerosis

The molecular basis of atherosclerosis is associated with excessive proliferation of vascular cells. Previous studies have suggested an inverse correlation between the expression of the growth suppressor p27Kip1 (p27) and cellular proliferation within human atherosclerotic tissue. However, no causal link between diminished p27 expression and atherogenesis has been established. We investigated the effect of p27 inactivation on diet‐induced atherogenesis. We find that p27‐deficient mice challenged with a high‐fat diet for 1 month remain normocholesterolemic and have essentially no visible atheromas. However, when generated in an apolipoprotein E‐null genetic background that leads to severe hypercholesterolemia in response to the atherogenic diet, deletion of p27 enhances arterial cell proliferation (~ fourfold) and accelerates atherogenesis (~sixfold) compared with apolipoprotein E‐defi‐cient mice with an intact p27 gene. Analysis of apoli‐poprotein E‐null mice bearing only one p27 allele inactivated reveals that a moderate decrease in p27 protein expression in the setting of hypercholesterolemia is sufficient to predispose to atherogenesis. Thus, our study establishes a molecular link between decreased p27 protein expression and atherogenesis in hypercholester‐olemic animals.

Short telomeres protect from diet-induced atherosclerosis in apolipoprotein E-null mice

By imposing a replicative defect in most somatic cells, gradual telomere attrition during aging is thought to progressively impair cellular function and viability and may contribute to age‐related disease. Immune cells play important roles in all phases of atherosclerosis, a multifactorial disease that prevails within the elderly. Because shorter telomeres have been found in circulating blood leukocytes of human patients with advanced coronary atherosclerosis, it has been suggested that telomere shortening may predispose the organism to atheroma development. In this study, we assessed the impact of telomere attrition on atherogenesis induced by dietary cholesterol in apolipoprotein E (apoE)‐deficient mice, a well‐established model of experimental atherosclerosis that recapitulates important aspects of the human disease. Our study shows that late‐generation mice doubly deficient in apoE and telomerase RNA experience telomere attrition and a substantial reduction of atherosclerosis compared with control mice with intact telomerase, in spite of sustained hypercholesterolemia in response to the atherogenic diet. Short telomeres impaired the proliferation of both lymphocytes and macrophages, an important step in atherosclerosis development. Therefore, telomere exhaustion resulting in replicative immunosenescence may serve as a mechanism for restricting atheroma progression.

Coordinate Control of Proliferation and Migration by the p27Kip1/Cyclin-Dependent Kinase/Retinoblastoma Pathway in Vascular Smooth Muscle Cells and Fibroblasts

Abstract— Previous studies have demonstrated a protective effect of the cyclin-dependent kinase (CDK) inhibitor p27Kip1 against atherosclerosis and restenosis, two disorders characterized by abundant proliferation and migration of vascular smooth muscle cells and adventitial fibroblasts. These therapeutic effects might result from p27Kip1-dependent suppression of both cell proliferation and migration. However, the interplay between cell growth and locomotion remains obscure. We show here that p27Kip1 inhibits cellular changes that normally occur during cell locomotion (eg, lamellipodia formation and reorganization of actin filaments and focal adhesions). Importantly, a p27Kip1 mutant lacking CDK inhibitory activity failed to inhibit vascular smooth muscle cell and fibroblast proliferation and migration. Moreover, a constitutively active mutant of the retinoblastoma protein (pRb) insensitive to CDK-dependent hyperphosphorylation inhibited both cell proliferation and migration. In contrast, inactivation of pRb by forced expression of the adenoviral oncogene E1A correlated with high proliferative and migratory activity. Collectively, these results suggest that cellular proliferation and migration are regulated in a coordinated manner by the p27Kip1/CDK/pRb pathway. These findings might have important implications for the development of novel therapeutic strategies targeting the fibroproliferative/migratory component of vascular occlusive disorders.

Distinct Regulation of Mitogen-activated Protein Kinases and p27Kip1 in Smooth Muscle Cells from Different Vascular Beds A POTENTIAL ROLE IN ESTABLISHING REGIONAL PHENOTYPIC VARIANCE

Excessive proliferation and migration of vascular smooth muscle cells (SMCs) participate in atherosclerotic plaque growth. In this study, we investigated whether SMCs from vessels with different atherogenicity exhibit distinct growth and migratory potential and investigated the underlying mechanisms. In fat-fed rabbits, we found increased cell proliferation and atheroma formation in the aortic arch versus the femoral artery. When examined in culture, SMCs isolated from the aortic arch (ASMCs) displayed a greater capacity for inducible proliferation and migration than paired cultures of femoral artery SMCs. Two lines of evidence suggested that distinct regulation of the growth suppressor p27Kip1(p27) contributes to establishing these phenotypic dissimilarities. First, p27 expression was comparably lower in ASMCs, which exhibited a higher fraction of p27 phosphorylated on Thr-187 and ubiquitinated. Second, forced p27 overexpression in ASMCs impaired their proliferative and migratory potential. We found that platelet-derived growth factor-BB-dependent induction of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway was comparably higher in ASMCs. Importantly, pharmacological inhibition of MAPKs increased p27 expression and attenuated ASMC proliferation and migration. In contrast, forced MAPK activation diminished p27 expression and markedly augmented femoral artery SMC proliferation and migration. We propose that intrinsic differences in the regulation of MAPKs and p27 play an important role in creating variance in the proliferative and migratory capacity of vascular SMCs, which might in turn contribute to establishing regional variability in atherogenicity.

A single-nucleotide polymorphism in the human p27kip1 gene (-838C>A) affects basal promoter activity and the risk of myocardial infarction

BackgroundExcessive proliferation of vascular smooth muscle cells and leukocytes within the artery wall is a major event in the development of atherosclerosis. The growth suppressor p27kip1 associates with several cyclin-dependent kinase/cyclin complexes, thereby abrogating their capacity to induce progression through the cell cycle. Recent studies have implicated p27kip1 in the control of neointimal hyperplasia. For instance, p27kip1 ablation in apolipoprotein-E-null mice enhanced arterial cell proliferation and accelerated atherogenesis induced by dietary cholesterol. Therefore, p27kip1 is a candidate gene to modify the risk of developing atherosclerosis and associated ischaemic events (i.e., myocardial infarction and stroke).ResultsIn this study we found three common single-nucleotide polymorphisms in the human p27kip1 gene (+326T>G [V109G], -79C>T, and -838C>A). The frequency of -838A carriers was significantly increased in myocardial infarction patients compared to healthy controls (odds ratio [OR] = 1.73, 95% confidence interval [95%CI] = 1.12–2.70). In addition, luciferase reporter constructs driven by the human p27kip1 gene promoter containing A at position -838 had decreased basal transcriptional activity when transiently transfected in Jurkat cells, compared with constructs bearing C in -838 (P = 0.04).ConclusionsThese data suggest that -838A is associated with reduced p27kip1 promoter activity and increased risk of myocardial infarction.

Increased early atherogenesis in young versus old hypercholesterolemic rabbits by a mechanism independent of arterial cell proliferation

We sought to determine the relative importance of aging and hypercholesterolemia on atherosclerosis. Although plasma cholesterol levels increased similarly in young and old rabbits fed an atherogenic diet for 2 months, aortic atherosclerotic lesions were more prominent in young animals. This finding was associated with an age‐dependent reduction in the DNA‐binding activity of the proinflammatory nuclear factor κB (NF‐κB) in aortic tissue. Atherosclerotic lesions consisted mostly of macrophages, which displayed a similar proliferative response in both age groups. Independently of the age, medial cell proliferation was low and increased as a function of intimal lesion size. Thus, higher atherogenicity in young rabbits exposed to extreme hypercholesterolemia compared to old counterparts is associated with higher activity of NF‐κB in the juvenile vessel wall without apparent age‐dependent changes in arterial cell proliferation.

Deficient p27 Phosphorylation at Serine 10 Increases Macrophage Foam Cell Formation and Aggravates Atherosclerosis Through a Proliferation-Independent Mechanism

Objective—Genetic ablation of the growth suppressor p27Kip1 (p27) in the mouse aggravates atherosclerosis coinciding with enhanced arterial cell proliferation. However, it is unknown whether molecular mechanisms that limit p27s protective function contribute to atherosclerosis development and whether p27 exerts proliferation-independent activities in the arterial wall. This study aims to provide insight into both questions by investigating the role in atherosclerosis of p27 phosphorylation at serine 10 (p27-phospho-Ser10), a major posttranslational modification of this protein. Methods and Results—Immunoblotting studies revealed a marked reduction in p27-phospho-Ser10 in atherosclerotic arteries from apolipoprotein E–null mice, and expression of the nonphosphorylatable mutant p27Ser10Ala, either global or restricted to bone marrow, accelerated atherosclerosis. p27Ser10Ala expression did not affect cell proliferation in early and advanced atheroma but activated RhoA/Rho-associated coiled-coil containing protein kinase (ROCK) signaling and promoted macrophage foam cell formation in a ROCK-dependent manner. Supporting the clinical relevance of these findings, human atherosclerotic coronary arteries exhibited a prominent reduction in p27-phospho-Ser10 and increased ezrin/radixin/moesin protein phosphorylation, a marker of RhoA/ROCK activation. Conclusion—Scarce phosphorylation of p27 at Ser10 is a hallmark of human and mouse atherosclerosis and promotes disease progression in mice. This proatherogenic effect is mediated by a proliferation-independent mechanism that involves augmented foam cell formation owing to increased RhoA/ROCK activity. These findings unveil a new atheroprotective action of p27 and identify p27-phospho-Ser10 as an attractive target for the treatment of atherosclerosis.

Podocalyxin-like protein 1 is a relevant marker for human c-kitpos cardiac stem cells

Cardiac progenitor cells (CPCs) from adult myocardium offer an alternative cell therapy approach for ischaemic heart disease. Improved clinical performance of CPCs in clinical trials requires a comprehensive definition of their biology and specific interactions with the environment. In this work we characterize specific human CPC surface markers and study some of their related functions. c‐kitpos human CPCs (hCPCs) were characterized for cell surface marker expression, pluripotency, early and late cardiac differentiation markers and therapeutic activity in a rat model of acute myocardial infarction. The results indicate that hCPCs are a mesenchymal stem cell (MSC)‐like population, with a similar immunoregulatory capacity. A partial hCPC membrane proteome was analysed by liquid chromatography–mass spectrometry/mass spectrometry and 36 proteins were identified. Several, including CD26, myoferlin and podocalyxin‐like protein 1 (PODXL), have been previously described in other stem‐cell systems. Suppression and overexpression analysis demonstrated that PODXL regulates hCPC activation, migration and differentiation; it also modulates their local immunoregulatory capacity. Therefore, hCPCs are a resident cardiac population that shares many features with hMSCs, including their capacity for local immunoregulation. Expression of PODXL appears to favour the immature state of hCPCs, while its downregulation facilitates their differentiation. Copyright

Inhibiting Cyclin-Dependent Kinase / Cyclin Activity for the Treatment of Cancer and Cardiovascular Disease

Excessive cell proliferation contributes to the pathobiology of human diseases with a high health and socio-economic impact, including cancer and vascular occlusive diseases (e. g., atherosclerosis, in-stent restenosis, transplant vasculopathy, and vessel bypass graft failure). Recent advances in the understanding of the molecular networks governing the hyperplastic growth of tumors and vascular obstructive neointimal lesions have provided new perspectives for preventive and therapeutic strategies against these disorders. Mammalian cell proliferation requires the activation of several cyclin-dependent protein kinases (CDKs). Postranslational activation of CDKs is a complex process that involves their association with regulatory subunits called cyclins. The activity of CDK/cyclin holoenzymes is negatively regulated through their interaction with members of the CDK family of inhibitory proteins (CKIs). Moreover, over fifty low molecular weight pharmacological CDK inhibitors that target the ATP-binding pocket of the catalytic site of CDKs have been identified. In this review, we will discuss the use of pharmacological and gene therapy strategies against CDK/cyclins in animal models and clinical trials of cancer and cardiovascular disease.