Chunyu Zeng

LincRNA-p21 Regulates Neointima Formation, Vascular Smooth Muscle Cell Proliferation, Apoptosis and Atherosclerosis by Enhancing p53 Activity

Background— Long noncoding RNAs (lncRNAs) have recently been implicated in many biological processes and diseases. Atherosclerosis is a major risk factor for cardiovascular disease. However, the functional role of lncRNAs in atherosclerosis is largely unknown. Methods and Results— We identified lincRNA-p21 as a key regulator of cell proliferation and apoptosis during atherosclerosis. The expression of lincRNA-p21 was dramatically downregulated in atherosclerotic plaques of ApoE−/− mice, an animal model for atherosclerosis. Through loss- and gain-of-function approaches, we showed that lincRNA-p21 represses cell proliferation and induces apoptosis in vascular smooth muscle cells and mouse mononuclear macrophage cells in vitro. Moreover, we found that inhibition of lincRNA-p21 results in neointimal hyperplasia in vivo in a carotid artery injury model. Genome-wide analysis revealed that lincRNA-p21 inhibition dysregulated many p53 targets. Furthermore, lincRNA-p21, a transcriptional target of p53, feeds back to enhance p53 transcriptional activity, at least in part, via binding to mouse double minute 2 (MDM2), an E3 ubiquitin-protein ligase. The association of lincRNA-p21 and MDM2 releases MDM2 repression of p53, enabling p53 to interact with p300 and to bind to the promoters/enhancers of its target genes. Finally, we show that lincRNA-p21 expression is decreased in patients with coronary artery disease. Conclusions— Our studies identify lincRNA-p21 as a novel regulator of cell proliferation and apoptosis and suggest that this lncRNA could serve as a therapeutic target to treat atherosclerosis and related cardiovascular disorders.

Perturbation of D1 Dopamine and AT1 Receptor Interaction in Spontaneously Hypertensive Rats

Abstract—The dopaminergic and renin-angiotensin systems interact to regulate blood pressure. Because this interaction may be perturbed in genetic hypertension, we studied D1 dopamine and AT1 angiotensin receptors in immortalized renal proximal tubule (RPT) and A10 aortic vascular smooth muscle cells. In normotensive Wistar-Kyoto (WKY) rats, the D1-like agonist fenoldopam increased D1 receptors but decreased AT1 receptors. These effects were blocked by the D1-like antagonist SCH 23390 (10−7 mol/L per 24 hours). In spontaneously hypertensive rat (SHR) RPT cells, fenoldopam also decreased AT1 receptors but no longer stimulated D1 receptor expression. Basal levels of AT1/D1 receptor coimmunoprecipitation were greater in WKY RPT cells (29±2 density units, DU) than in SHR RPT cells (21±2 DU, n=7 per group, P <0.05). The coimmunoprecipitation of D1 and AT1 receptors was increased by fenoldopam (10−7 mol/L per 24 hours) in WKY RPT cells but decreased in SHR RPT cells. The effects of fenoldopam in RPT cells from WKY rats were similar in aortic vascular smooth muscle cells from normotensive BD IX rats, that is, fenoldopam decreased AT1 receptors and increased D1 receptors. Our studies show differential regulation of the expression of D1 and AT1 receptors in RPT cells from WKY and SHR. This regulation and D1/AT1 receptor interaction are different in RPT cells of WKY and SHR. An altered interaction of D1 and AT1 receptors may play a role in the impaired sodium excretion and enhanced vasoconstriction in hypertension.

The dopaminergic system in hypertension

Dopamine plays an important role in the pathogenesis of hypertension by regulating epithelial sodium transport, vascular smooth muscle contractility and production of reactive oxygen species and by interacting with the renin-angiotensin and sympathetic nervous systems. Dopamine receptors are classified into D(1)-like (D(1) and D(5)) and D(2)-like (D(2), D(3) and D(4)) subtypes based on their structure and pharmacology. Each of the dopamine receptor subtypes participates in the regulation of blood pressure by mechanisms specific for the subtype. Some receptors regulate blood pressure by influencing the central and/or peripheral nervous system; others influence epithelial transport and regulate the secretion and receptors of several humoral agents. This review summarizes the physiology of the different dopamine receptors in the regulation of blood pressure, and the relationship between dopamine receptor subtypes and hypertension.

Activation of D3 Dopamine Receptor Decreases Angiotensin II Type 1 Receptor Expression in Rat Renal Proximal Tubule Cells

The dopaminergic and renin angiotensin systems interact to regulate blood pressure. Disruption of the D3 dopamine receptor gene in mice produces renin-dependent hypertension. In rats, D2-like receptors reduce angiotensin II binding sites in renal proximal tubules (RPTs). Because the major D2-like receptor in RPTs is the D3 receptor, we examined whether D3 receptors regulate angiotensin II type 1 (AT1) receptors in rat RPT cells. The effect of D3 receptors on AT1 receptors was studied in vitro and in vivo. The D3 receptor agonist PD128907 decreased AT1 receptor protein and mRNA in WKY RPT cells and increased it in SHR cells. PD128907 increased D3 receptors in WKY cells but had no effect in SHR cells. D3/AT1 receptors colocalized in RPT cells; D3 receptor stimulation decreased the percent amount of D3 receptors that coimmunoprecipitated with AT1 receptors to a greater extent in WKY than in SHR cells. However, D3 receptor stimulation did not change the percent amount of AT1 receptors that coimmunoprecipitated with D3 receptors in WKY cells and markedly decreased the coimmunoprecipitation in SHR cells. The D3 receptor also regulated the AT1 receptor in vivo because AT1 receptor expression was increased in kidneys of D3 receptor–null mice compared with wild type littermates. D3 receptors may regulate AT1 receptor function by direct interaction with and regulation of AT1 receptor expression. One mechanism of hypertension may be related to increased renal expression of AT1 receptors due decreased D3 receptor regulation.

Interaction of Angiotensin II Type 1 and D5 Dopamine Receptors in Renal Proximal Tubule Cells

Angiotensin II type 1 (AT1) receptor and D1 and D3 dopamine receptors directly interact in renal proximal tubule (RPT) cells from normotensive Wistar-Kyoto rats (WKY). There is indirect evidence for a D5 and AT1 receptor interaction in WKY and spontaneously hypertensive rats (SHR). Therefore, we sought direct evidence of an interaction between AT1 and D5 receptors in RPT cells. D5 and AT1 receptors colocalized in WKY cells. Angiotensin II decreased D5 receptors in WKY cells in a time- and concentration-dependent manner (EC50=2.7×10−9 M; t1/2=4.9 hours), effects that were blocked by an AT1 receptor antagonist (losartan). In SHR, angiotensin II (10−8 M/24 hours) also decreased D5 receptors (0.96±0.08 versus 0.72±0.08; n=12) and to the same degree as in WKY cells (1.44±0.07 versus 0.92±0.08). However, basal D5 receptors were decreased in SHR RPT cells (SHR 0.96±0.08; WKY 1.44±0.07; n=12 per strain; P<0.05) and renal brush border membranes of SHR compared with WKY (SHR 0.54±0.16 versus WKY 1.46±0.10; n=5 per strain; P<0.05). Angiotensin II decreased AT1 receptor expression in WKY (1.00±0.04 versus 0.72±0.08; n=8; P<0.05) but increased it in SHR (0.96±0.04 versus 1.32±0.08; n=8; P<0.05). AT1 and D5 receptors also interacted in vivo; renal D5 receptor protein was higher in mice lacking the AT1A receptor (AT1A−/−; 1.61±0.31; n=6) than in wild-type littermates used as controls (AT1A+/+; 0.81±0.08; n=6; P<0.05), and renal cortical AT1 receptor protein was higher in D5 receptor null mice than in wild-type littermates (1.18±0.08 versus 0.84±0.07; n=4; P<0.05). We conclude that D5 and AT1 receptors interact with each other. Altered interactions between AT1 and dopamine receptors may play a role in the pathogenesis of hypertension.

Angiotensin II Regulation of AT1 and D3 Dopamine Receptors in Renal Proximal Tubule Cells of SHR

Abstract—Dopamine and angiotensin II negatively interact to regulate sodium excretion and blood pressure. D3 dopamine receptors downregulate angiotensin type 1 (AT1) receptors in renal proximal tubule cells from normotensive Wistar-Kyoto rats. We determined whether AT1 receptors regulate D3 receptors and whether the regulation is different in cultured renal proximal tubule cells from normotensive and spontaneously hypertensive rats. Angiotensin II (10−8M/24 hours) decreased D3 receptors in both normotensive (control, 36±3; angiotensin II, 24±3 U) and hypertensive (control, 30±3; angiotensin II, 11±3 U; n=9 per group) rats; effects that were blocked by the AT1 receptor antagonist, losartan (10−8M/24 hours). However, the reduction in D3 expression was greater in hypertensive (60±10%) than in normotensive rats (32±9%). In normotensive rats, angiotensin II (10−8M/24hr) also decreased AT1 receptors. In contrast, in cells from hypertensive rats, angiotensin II increased AT1 receptors. AT1 and D3 receptors co-immunoprecipitated in renal proximal tubule cells from both strains. Angiotensin II decreased D3/AT1 receptor co-immunoprecipitation similarly in both rat strains, but basal D3/AT1 co-immunoprecipitation was 6 times higher in normotensive than in hypertensive rats. Therefore, AT1 and D3 receptor interaction is qualitatively and quantitatively different between normotensive and hypertensive rats; angiotensin II decreases AT1 expression in normotensive but increases it in hypertensive rats. In addition, angiotensin II decreases D3 expression to a greater extent in hypertensive than in normotensive rats. Aberrant interactions between D3 and AT1 receptors may play a role in the pathogenesis of hypertension.

Extracellular vesicle-mediated transfer of donor genomic DNA to recipient cells is a novel mechanism for genetic influence between cells

Extracellular vesicles (EVs) carry signals within or at their limiting membranes, providing a mechanism by which cells can exchange more complex information than what was previously thought. In addition to mRNAs and microRNAs, there are DNA fragments in EVs. Solexa sequencing indicated the presence of at least 16434 genomic DNA (gDNA) fragments in the EVs from human plasma. Immunofluorescence study showed direct evidence that acridine orange-stained EV DNAs could be transferred into the cells and localize to and inside the nuclear membrane. However, whether the transferred EV DNAs are functional or not is not clear. We found that EV gDNAs could be homologously or heterologously transferred from donor cells to recipient cells, and increase gDNA-coding mRNA, protein expression, and function (e.g. AT1 receptor). An endogenous promoter of the AT1 receptor, NF-κB, could be recruited to the transferred DNAs in the nucleus, and increase the transcription of AT1 receptor in the recipient cells. Moreover, the transferred EV gDNAs have pathophysiological significance. BCR/ABL hybrid gene, involved in the pathogenesis of chronic myeloid leukemia, could be transferred from K562 EVs to HEK293 cells or neutrophils. Our present study shows that the gDNAs transferred from EVs to cells have physiological significance, not only to increase the gDNA-coding mRNA and protein levels, but also to influence function in recipient cells.

The relative contribution of paracine effect versus direct differentiation on adipose-derived stem cell transplantation mediated cardiac repair.

Background Recent studies have demonstrated that transplantation of adipose-derived stem cell (ADSC) can improve cardiac function in animal models of myocardial infarction (MI). However, the mechanisms underlying the beneficial effect are not fully understood. In this study, we characterized the paracrine effect of transplanted ADSC and investigated its relative importance versus direct differentiation in ADSC transplantation mediated cardiac repair. Methodology/Principal Findings MI was experimentally induced in mice by ligation of the left anterior descending coronary artery. Either human ADSC, conditioned medium (CM) collected from the same amount of ADSC or control medium was injected into the peri-infarct region immediately after MI. Compared with the control group, both ADSC and ADSC-CM significantly reduced myocardial infarct size and improved cardiac function. The therapeutic efficacy of ADSC was moderately superior to ADSC-CM. ADSC-CM significantly reduced cardiomyocyte apoptosis in the infarct border zone, to a similar degree with ADSC treatment. ADSC enhanced angiogenesis in the infarct border zone, but to a stronger degree than that seen in the ADSC-CM treatment. ADSC was able to differentiate to endothelial cell and smooth muscle cell in post-MI heart; these ADSC-derived vascular cells amount to about 9% of the enhanced angiogenesis. No cardiomyocyte differentiated from ADSC was found. Conclusions ADSC-CM is sufficient to improve cardiac function of infarcted hearts. The therapeutic function of ADSC transplantation is mainly induced by paracrine-mediated cardioprotection and angiogenesis, while ADSC differentiation contributes a minor benefit by being involved in angiogenesis. Highlights 1 ADSC-CM is sufficient to exert a therapeutic potential. 2. ADSC was able to differentiate to vascular cells but not cardiomyocyte. 3. ADSC derived vascular cells amount to about 9% of the enhanced angiogenesis. 4. Paracrine effect is the major mechanism of ADSC therapeutic function for MI.

Amelioration of Genetic Hypertension by Suppression of Renal G Protein-Coupled Receptor Kinase Type 4 Expression

Abnormalities in D1 dopamine receptor function in the kidney are present in some types of human essential and rodent genetic hypertension. We hypothesize that increased activity of G protein–coupled receptor kinase type 4 (GRK4) causes the impaired renal D1 receptor function in hypertension. We measured renal GRK4 and D1 and serine-phosphorylated D1 receptors and determined the effect of decreasing renal GRK4 protein by the chronic renal cortical interstitial infusion (4 weeks) of GRK4 antisense oligodeoxynucleotides (As-Odns) in conscious- uninephrectomized spontaneously hypertensive rats (SHRs) and their normotensive controls, Wistar–Kyoto (WKY) rats. Basal GRK4 expression and serine-phosphorylated D1 receptors were ≈90% higher in SHRs than in WKY rats and were decreased to a greater extent in SHRs than in WKY rats with GRK4 As-Odns treatment. Basal renal D1 receptor protein was similar in both rat strains. GRK4 As-Odns, but not scrambled oligodeoxynucleotides, increased sodium excretion and urine volume, attenuated the increase in arterial blood pressure with age, and decreased protein excretion in SHRs, effects that were not observed in WKY rats. These studies provide direct evidence of a crucial role of renal GRK4 in the D1 receptor control of sodium excretion and blood pressure in genetic hypertension.

Dopamine D1 Receptor Augmentation of D3 Receptor Action in Rat Aortic or Mesenteric Vascular Smooth Muscles

Abstract—Dopamine is an important modulator of blood pressure, in part, by regulating vascular resistance. To test the hypothesis that D1 and D3 receptors interact in vascular smooth muscle cells, we studied A10 cells, a rat aortic smooth muscle cell line, and rat mesenteric arteries that express both dopamine receptor subtypes. Fenoldopam, a D1-like receptor agonist, increased both D1 and D3 receptor protein in a time-dependent and a concentration-dependent manner in A10 cells. The effect of fenoldopam was specific because a D1-like receptor antagonist, SCH23390 (10−7 M/24 h), completely blocked the stimulatory effect of fenoldopam (10−7 M/24 h) (D3 receptor: control=21±1 density units [DU]); SCH23390=23±2 DU; fenoldopam=33±2 DU; fenoldopam+SCH23390=23±2 DU; n=10). D1 and D3 receptors physically interacted with each other because fenoldopam (10−7 M/24 h) increased D1/D3 receptor coimmunoprecipitation (35±5 versus 65±5 DU; n=8). A D3 receptor agonist, PD128907, relaxed mesenteric arterial rings independent of the endothelium, effects that were blocked by a D3 receptor antagonist, U99194A. Costimulation of D1 and D3 receptors led to additive vasorelaxation. We conclude that the D1 receptor regulates the D3 receptor by physical interaction and receptor expression. D1 receptor stimulation augments D3 receptor vasorelaxant effects. An interaction of D1 and D3 receptors may be involved in the regulation of blood pressure.