Jianzhong Shen

ADP Stimulates Human Endothelial Cell Migration via P2Y1 Nucleotide Receptor–Mediated Mitogen-Activated Protein Kinase Pathways

Extensive research on the role of ADP in platelet activation led to the design of new anti-thrombotic drugs, such as clopidogrel (Plavix; sanofi-aventis); however, very little is known about the ADP-preferring nucleotide receptors (P2Y1, P2Y12, and P2Y13) in endothelium. Here, we show that ADP stimulates migration of cultured human umbilical vein endothelial cells (HUVECs) in both Boyden chamber and in vitro wound repair assays. This promigratory effect was mimicked by 2-MeSADP, but not by AMP, and was inhibited by MRS2179 (P2Y1 receptor antagonist) but not by AR-C69931MX (P2Y12/13 receptor antagonist). RT-PCR revealed abundant P2Y1, barely detectable P2Y12, and absent P2Y13 receptor message in these cells. In addition, both ADP and 2-MeSADP, but not AMP, activated the mitogen-activated protein kinase pathways as evidenced by increased phosphorylation of extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK), and p38 kinase. ADP also stimulated phosphorylation of p90RSK, a downstream substrate of phosphorylated ERK1/2, and induced phosphorylation of such transcription factors downstream of the JNK and p38 pathways as c-Jun and activating transcription factor-2. These signaling events were inhibited by MRS2179 but not by AR-C69931MX. Furthermore, blockade of the ERK or JNK pathways by U0126 and SP600125, respectively, abolished ADP- and 2-MeSADP-stimulated HUVEC migration. However, inhibition of the p38 pathway by SB203580 partially suppressed ADP- and 2-MeSADP-induced HUVEC migration. We conclude that ADP promotes human endothelial cell migration by activating P2Y1 receptor-mediated MAPK pathways, possibly contributing to reendothelialization and angiogenesis after vascular injury.

Induction of C-X-C Chemokine Receptor Type 7 (CXCR7) Switches Stromal Cell-derived Factor-1 (SDF-1) Signaling and Phagocytic Activity in Macrophages Linked to Atherosclerosis

Background: Previously, whether the new SDF-1 receptor CXCR7 plays a role in macrophages linked to disease was unknown. Results: During macrophage differentiation, CXCR7 is up-regulated, detected in mouse atherosclerotic plaques, and mediates pro-phagocytic activity via JNK and p38 pathways. Conclusion: CXCR7 is a functional SDF-1 receptor in macrophages. Significance: Macrophage CXCR7 might be a new therapeutic target for atherosclerosis. The discovery of CXCR7 as a new receptor for SDF-1 places many previously described SDF-1 functions attributed to CXCR4 in question, though whether CXCR7 acts as a signaling or “decoy” receptor has been in debate. It is known that CXCR7 is not expressed in normal blood leukocytes; however, the potential role of leukocyte CXCR7 in disease states has not been addressed. The aim of this study was to determine the expression and function of macrophage CXCR7 linked to atherosclerosis. Here, we show that CXCR7 was detected in macrophage-positive area of aortic atheroma of ApoE-null mice, but not in healthy aorta. During monocyte differentiation to macrophages, CXCR7 was up-regulated at mRNA and protein levels, with more expression in M1 than in M2 phenotype. In addition, CXCR7 induction was associated with a SDF-1 signaling switch from the pro-survival ERK and AKT pathways in monocytes to the pro-inflammatory JNK and p38 pathways in macrophages. The latter effect was mimicked by a CXCR7-selective agonist TC14012 and abolished by siRNA knockdown of CXCR7. Furthermore, CXCR7 activation increased macrophage phagocytic activity, which was suppressed by CXCR7 siRNA silencing or by inhibiting either the JNK or p38 pathways, but was not affected by blocking CXCR4. Finally, activation of CXCR7 by I-TAC showed a similar signaling and phagocytic activity in macrophages with no detectable CXCR3. We conclude that CXCR7 is induced during monocyte-to-macrophage differentiation, which is required for SDF-1 and I-TAC signaling to JNK and p38 pathways, leading to enhanced macrophage phagocytosis, thus possibly contributing to atherogenesis.

Cell-Signaling Evidence for Adenosine Stimulation of Coronary Smooth Muscle Proliferation via the A1 Adenosine Receptor

For decades, it has been thought that adenosine is exclusively antimitogenic on vascular smooth muscles via the A2-type adenosine receptor. Recently, we have demonstrated that adenosine stimulates proliferation of porcine coronary artery smooth muscle cells (CASMC) through the A1 adenosine receptor. However, the cell-signaling mechanisms underlying A1 receptor–mediated CASMC proliferation in response to adenosine have not been defined. Here, we show that in cultured CASMC, adenosine stimulates phosphorylation of extracellular signal–regulated kinase (ERK), Jun N-terminal kinase (JNK), and AKT in a concentration- and time-dependent manner. This effect is fully mimicked by NECA (nonselective agonist), largely mimicked by CCPA (A1-selective agonist), weakly mimicked by 2-Cl-IB-MECA (A3-selective agonist), but not by CGS21680 (A2A-selective agonist), indicating that adenosine signals strongly via the A1 receptor to these mitogenic signaling pathways. This interpretation is supported by the finding that adenosine- and CCPA-induced phosphorylation of ERK, JNK, and AKT are inhibited by pertussis toxin (inactivator of Gi proteins) and by DPCPX (A1-selective antagonist), but not by SCH58261, MRS1706, and VUF5574 (A2A-, A2B-, and A3-selective antagonists, respectively). In addition, adenosine- and CCPA-induced phosphorylation of ERK, JNK, and AKT is inhibited, respectively, by U0126, PD98059 (mitogen-activated protein kinase kinase inhibitors), SP600125 (JNK kinase inhibitor), and wortmannin (phosphatidylinositol 3-kinase inhibitor). Furthermore, these kinase inhibitors abolish or diminish adenosine- and CCPA-induced increases in the rate of cellular DNA synthesis, bromodeoxyuridine incorporation, protein synthesis, and cell number. We conclude that adenosine activates the ERK, JNK, and phosphatidylinositol 3-kinase/AKT pathways primarily through the A1 receptor, leading to CASMC mitogenesis.

Corin mutation R539C from hypertensive patients impairs zymogen activation and generates an inactive alternative ectodomain fragment.

Background: Corin is a membrane serine protease that activates natriuretic peptides in the heart. Results: The corin mutant R539C identified in hypertensive patients has impaired zymogen activation and altered ectodomain shedding. Conclusion: The mutation alters corin protein structure and reduces corin activity. Significance: Human CORIN gene mutations causing impaired corin activity may be an underlying mechanism in hypertension. Corin is a cardiac transmembrane serine protease that regulates blood pressure by activating natriuretic peptides. Corin variants have been associated with African Americans with hypertension and heart disease. Here, we report a new mutation in exon 12 of the CORIN gene identified in a family of patients with hypertension. The mutation resulted in R539C substitution in the Fz2 (Frizzled-2) domain of the corin propeptide region. We expressed and characterized the corin R539C mutant in HEK293 cells. As determined by Western blot analysis, the R539C mutation did not alter corin expression in transfected cells but impaired corin zymogen activation. In a pro-atrial natriuretic peptide processing assay, the corin mutant had reduced activity and exhibited a dominant-negative effect on wild-type corin. In addition, the R539C mutation altered corin ectodomain shedding, producing an alternative ∼75-kDa fragment that was biologically inactive. Using protease inhibitors and the catalytically inactive corin mutant S985A, we showed that the ∼75-kDa fragment was generated by corin autocleavage. We constructed a series of mutants by replacing single or double Arg residues in the corin propeptide and identified Arg-530 in the Fz2 domain as the alternative autocleavage site. Our results show that the corin mutation R539C identified in hypertensive patients impairs corin zymogen activation and causes an alternative autocleavage that reduces corin activity. These data support that human CORIN gene mutations causing impaired corin activity may be an underlying mechanism in hypertension.

Novel Mitogenic Effect of Adenosine on Coronary Artery Smooth Muscle Cells Role for the A1 Adenosine Receptor

Adenosine is a vascular endothelial cell mitogen, but anti-mitogenic for aortic smooth muscle cells and fibroblasts when acting via the A2B adenosine receptor. However, we show that adenosine increases porcine coronary artery smooth muscle cell (CASMC) number, cellular DNA content, protein synthesis, and PCNA staining. RT-PCR analysis indicates that porcine CASMC express A1, A2A, A3, and barely detectable levels of A2B receptor mRNAs. The mitogenic effect of adenosine is mimicked by NECA, CCPA, and R-PIA, but not by CGS21680 and 2-Cl-IB-MECA, and is inhibited by DPCPX, indicating a prominent role for the A1 receptor. This interpretation is supported by the finding that adenosine- and CCPA-induced DNA synthesis is significantly inhibited by pertussis toxin, but substantially potentiated by PD81723, an allosteric enhancer of the A1 receptor. When a cDNA encoding the porcine A1 receptor was cloned and expressed in COS-1 cells, A1 receptor pharmacology is confirmed. Anti-sense oligonucleotides to the cloned sequence dramatically suppress the mitogenic effect of adenosine and CCPA. Conversely, over-expression of the cloned A1 receptor in CASMC increases adenosine- and CCPA-induced DNA synthesis. Furthermore, stimulation with adenosine or CCPA of intact coronary arteries in an organ culture model of vascular disease increases cellular DNA synthesis, which was abolished by DPCPX. We conclude that adenosine acts as a novel mitogen in porcine CASMC that express the A1 adenosine receptor, possibly contributing to the development of coronary artery disease.

Lack of Mitogen-Activated Protein Kinase Phosphatase-1 Protects ApoE-Null Mice Against Atherosclerosis

Rationale: Multiple protein kinases have been implicated in cardiovascular disease; however, little is known about the role of their counterparts: the protein phosphatases. Objective: To test the hypothesis that mitogen-activated protein kinase phosphatase (MKP)-1 is actively involved in atherogenesis. Methods and Results: Mice with homozygous deficiency in MKP-1 (MKP-1−/−) were bred with apolipoprotein (Apo)E-deficient mice (ApoE−/−) and the 3 MKP-1 genotypes (MKP-1+/+/ApoE−/−; MKP-1+/−/ApoE−/− and MKP-1−/−/ApoE−/−) were maintained on a normal chow diet for 16 weeks. The 3 groups of mice exhibited similar body weight and serum lipid profiles; however, both MKP-1+/− and MKP-1−/− mice had significantly less aortic root atherosclerotic lesion formation than MKP-1+/+ mice. Less en face lesion was observed in 8-month-old MKP-1−/− mice. The reduction in atherosclerosis was accompanied by decreased plasma levels of interleukin-1&agr; and tumor necrosis factor &agr;, and preceded by increased antiinflammatory cytokine interleukin-10. In addition, MKP-1–null mice had higher levels of plasma stromal cell–derived factor-1a, which negatively correlated with atherosclerotic lesion size. Immuno-histochemical analysis revealed that MKP-1 expression was enriched in macrophage-rich areas versus smooth muscle cell regions of the atheroma. Furthermore, macrophages isolated from MKP-1–null mice showed dramatic defects in their spreading/migration and impairment in extracellular signal-regulated kinase, but not c-Jun N-terminal kinase and p38, pathway activation. In line with this, MKP-1–null atheroma exhibited less macrophage content. Finally, transplantation of MKP-1–intact bone marrow into MKP-1–null mice fully rescued the wild-type atherosclerotic phenotype. Conclusion: These findings demonstrate that chronic deficiency of MKP-1 leads to decreased atherosclerosis via mechanisms involving impaired macrophage migration and defective extracellular signal-regulated kinase signaling.

The P2Y2 Nucleotide Receptor Mediates Tissue Factor Expression in Human Coronary Artery Endothelial Cells

The discovery of the role of P2Y12 receptor in platelet aggregation leads to a new anti-thrombotic drug Plavix; however, little is known about non-platelet P2Y receptors in thrombosis. This study tested the hypothesis that endothelial P2Y receptor(s) mediates up-regulation of tissue factor (TF), the initiator of coagulation cascade. Stimulation of human coronary artery endothelial cells (HCAEC) by UTP/ATP increased the mRNA level of TF but not of its counterpart-tissue factor pathway inhibitor, which was accompanied by up-regulation of TF protein and cell surface activity. RT-PCR revealed a selective expression of P2Y2 and P2Y11 receptors in HCAEC. Consistent with this, TF up-regulation was inhibited by suramin or by siRNA silencing of P2Y2 receptor, but not by NF-157, a P2Y11-selective antagonist, suggesting a role for the P2Y2 receptor. In addition, P2Y2 receptor activated ERK1/2, JNK, and p38 MAPK pathways without affecting the positive NF-κB and negative AKT regulatory pathways of TF expression. Furthermore, TF up-regulation was abolished or partially suppressed by inhibition of p38 or JNK but not ERK1/2. Interestingly, blockade of the PLC/Ca2+ pathway did not affect P2Y2 receptor activation of p38, JNK, and TF induction. However, blockade of Src kinase reduced phosphorylation of p38 but not JNK, eliminating TF induction. In contrast, inhibition of Rho kinase reduced phosphorylation of JNK but not p38, decreasing TF expression. These findings demonstrate that P2Y2 receptor mediates TF expression in HCAEC through new mechanisms involving Src/p38 and Rho/JNK pathways, possibly contributing to a pro-thrombotic status after vascular injury.

Atorvastatin inhibits CXCR7 induction to reduce macrophage migration

We have recently reported that CXCR7, the alternate high affinity SDF-1 receptor, is induced during monocyte-to-macrophage differentiation, leading to increased macrophage phagocytosis linked to atherosclerosis. Statins, the most widely used medications for atherosclerosis, were shown to have pleiotropic beneficial effects independent of their cholesterol-lowering activity. This study aimed to determine whether induction of CXCR7 during macrophage differentiation is inhibited by statins and its significance on macrophage physiology. Here we show for the first time that atorvastatin dose-dependently inhibited CXCR7 mRNA and protein expression in THP-1 macrophages, without affecting the other SDF-1 receptor, CXCR4. Pharmacotherapy relevant dose of atorvastatin affected neither cell viability nor macrophage differentiation. Suppression of CXCR7 expression was completely reversed by supplementation with mevalonate. Inhibition of squalene synthase, the enzyme committed to cholesterol biosynthesis, also decreased CXCR7 induction, albeit not as efficacious as atorvastatin. However, the geranylgeranyl transferase inhibitor, GGTI-286, the farnesyl transferase inhibitor, FTI-276, and the Rho kinase inhibitor, Y-27632, all failed to mimic the effect of atorvastatin, suggesting that the protein prenylation pathways are not critical for atorvastatin inhibition of CXCR7 induction. Interestingly, the dramatic effect of atorvastatin was only partially mimicked by other statins including pravastatin, fluvastatin, mevastatin, and simvastatin. Furthermore, activation of CXCR7 by SDF-1, TC14012, or I-TAC all prompted macrophage migration, which was significantly suppressed by atorvastatin treatment, but not by the CXCR4 antagonist. We conclude that atorvastatin modulates macrophage migration by down-regulating CXCR7 expression, suggesting a new CXCR7-dependent mechanism of atorvastatin to benefit atherosclerosis treatment beyond its lipid lowering effect.

Adenosine Prompts the Heart to Recruit Endothelial Progenitors

See related article, pages 356–363 Adenosine has a long history of involvement in cardiac function. It was first purified from mammalian heart homogenates in 1929 by Drury and Szent-Gyorgyi,1 who reported a “suppressive” action of adenosine on the heart, including decreased heart beat rate and perfusion pressure,1 now termed bradycardia and coronary artery vasodilation, respectively. Based on a variety of experimental results from several laboratories, Berne et al proposed in the early 1960s that adenosine plays an important role in the adjustment of blood flow to the metabolic requirements of organs, including the heart, brain, and skeletal muscle.2 Numerous studies now support the hypothesis that adenosine can be directly released or generated from released ATP/ADP in the heart during ischemia, causing increased oxygen and nutrition supply by inducing coronary artery relaxation.3 Although adenosine exhibits reuptake via specific adenosine transporters, many, if not all, of the biological effects of adenosine are believed to be mediated by adenosine receptors (ARs), of which 4 subtypes (A1R, A2AR, A2BR, and A3R) have been cloned and pharmacologically characterized.4 The diverse physiological functions mediated by the different AR subtypes, particularly in the modulation of the cardiovascular system, have been confirmed by genetically engineered mice.4 Null mice have been generated for each of the AR subtypes, and none of the 4 ARs plays a critical role during development.4,5 Although each AR protein had been thought to exist in the cell membrane as a monomer, recent studies with recombinant expression systems have revealed that some of the AR subtypes heterodimerize with other G-protein coupled receptors. For example, A1R has been shown to heterodimerize with …

Deacetylase SIRT6 deaccelerates endothelial senescence.

This editorial refers to ‘SIRT6 protects human endothelial cells from DNA damage, telomere dysfunction, and senescence’ by A. Cardus et al ., pp. 571–579, this issue. Vascular endothelial homeostasis is critical to health and is a significant factor in many human diseases. It is established that endothelial cell (EC) dysfunction is one of the earliest pathological steps in the initiation and progression of vascular diseases such as atherosclerosis and vascular remodelling.1–3 How to maintain a healthy or repair an injured endothelial lining of the vasculature has been a scientific mystery and a clinical challenge. In general, two theories have been proposed. On the one hand, it is thought that during their turnover, injured ECs, due to senescence, ageing, or other pathological challenges, might be replaced by circulating EC progenitors that can repopulate the damaged endothelium. Although this hypothesis has undergone rigorous investigation and achieved some promising initial results,4,5 recent studies demonstrated that circulating EC progenitors do not contribute to the regeneration of a damaged endothelium, thus significantly reducing this theorys promise in the field.6 On the other hand, healing damaged EC areas with adjacent mature ECs through their migration and proliferation is still a viable theory.7 In order to achieve this ‘ in situ healing’, however, the pre-existing mature ECs in the vasculature may have to maintain a ‘threshold’ of healthy …