Hua Qing

Epigenetic Regulation of Vascular Smooth Muscle Cell Proliferation and Neointima Formation by Histone Deacetylase Inhibition

Objective—Proliferation of smooth muscle cells (SMC) in response to vascular injury is central to neointimal vascular remodeling. There is accumulating evidence that histone acetylation constitutes a major epigenetic modification for the transcriptional control of proliferative gene expression; however, the physiological role of histone acetylation for proliferative vascular disease remains elusive. Methods and Results—In the present study, we investigated the role of histone deacetylase (HDAC) inhibition in SMC proliferation and neointimal remodeling. We demonstrate that mitogens induce transcription of HDAC 1, 2, and 3 in SMC. Short interfering RNA–mediated knockdown of either HDAC 1, 2, or 3 and pharmacological inhibition of HDAC prevented mitogen-induced SMC proliferation. The mechanisms underlying this reduction of SMC proliferation by HDAC inhibition involve a growth arrest in the G1 phase of the cell cycle that is due to an inhibition of retinoblastoma protein phosphorylation. HDAC inhibition resulted in a transcriptional and posttranscriptional regulation of the cyclin-dependent kinase inhibitors p21Cip1 and p27Kip. Furthermore, HDAC inhibition repressed mitogen-induced cyclin D1 mRNA expression and cyclin D1 promoter activity. As a result of this differential cell cycle-regulatory gene expression by HDAC inhibition, the retinoblastoma protein retains a transcriptional repression of its downstream target genes required for S phase entry. Finally, we provide evidence that these observations are applicable in vivo by demonstrating that HDAC inhibition decreased neointima formation and expression of cyclin D1 in a murine model of vascular injury. Conclusion—These findings identify HDAC as a critical component of a transcriptional cascade regulating SMC proliferation and suggest that HDAC might play a pivotal role in the development of proliferative vascular diseases, including atherosclerosis and in-stent restenosis.

Oxidative Stress Accumulates in Adipose Tissue during Aging and Inhibits Adipogenesis

Aging constitutes a major independent risk factor for the development of type 2 diabetes and is accompanied by insulin resistance and adipose tissue dysfunction. One of the most important factors implicitly linked to aging and age-related chronic diseases is the accumulation of oxidative stress. However, the effect of increased oxidative stress on adipose tissue biology remains elusive. In this study, we demonstrate that aging in mice results in a loss of fat mass and the accumulation of oxidative stress in adipose tissue. In vitro, increased oxidative stress through glutathione depletion inhibits preadipocyte differentiation. This inhibition of adipogenesis is at least in part the result of reduced cell proliferation and an inhibition of G1→S-phase transition during the initial mitotic clonal expansion of the adipocyte differentiation process. While phosphorylation of the retinoblastoma protein (Rb) by cyclin/cdk complexes remains unaffected, oxidative stress decreases the expression of S-phase genes downstream of Rb. This silencing of S phase gene expression by increased oxidative stress is mediated through a transcriptional mechanism involving the inhibition of E2F recruitment and transactivation of its target promoters. Collectively, these data demonstrate a previously unrecognized role of oxidative stress in the regulation of adipogenesis which may contribute to age-associated adipose tissue dysfunction.

Serum pigment epithelium-derived factor is elevated in women with polycystic ovary syndrome and correlates with insulin resistance.

CONTEXT Serum pigment epithelium-derived factor (PEDF) is highly expressed in adipose tissue and plays an important role in insulin resistance (IR). However, there are no data on serum PEDF levels and their relationship with IR in polycystic ovary syndrome (PCOS) women. OBJECTIVE To quantitate serum PEDF levels and examine their relationship with IR in women with PCOS. PARTICIPANTS AND DESIGN Ninety-six PCOS women and 63 healthy age-matched controls were recruited. Ninety-six PCOS women and 20 controls underwent hyperinsulinemic-euglycemic clamp to assess their insulin sensitivity, which was expressed as M value. IR was also estimated by homeostasis model assessment 2 (HOMA2-IR). SETTING The study was performed at a clinical research center. RESULTS PCOS women had lower M value and higher HOMA2-IR as compared with controls. Serum PEDF levels were much higher in PCOS women than in controls (5.45 ± 1.85 vs. 3.97 ± 0.98 μg/ml, P < 0.01). Spearman correlation analysis showed that in PCOS women, PEDF positively correlated with body mass index, waist circumference, HOMA2-IR, triglycerides, total cholesterol, low-density lipoprotein cholesterol, and systolic blood pressure and negatively correlated with M value and high-density lipoprotein cholesterol. Multiple linear regression analysis revealed that in PCOS women, after adjustment for body mass index, systolic blood pressure, and serum lipids (triglycerides, total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol), PEDF was still associated with M value or HOMA2-IR. CONCLUSIONS The serum PEDF level is elevated in women with PCOS and is associated with IR. PEDF may play a role in the pathogenesis of IR in PCOS.

Glutathione Depletion Prevents Diet-Induced Obesity and Enhances Insulin Sensitivity

Excessive accumulation of reactive oxygen species (ROS) in adipose tissue has been implicated in the development of insulin resistance and type 2 diabetes. However, emerging evidence suggests a physiologic role of ROS in cellular signaling and insulin sensitivity. In this study, we demonstrate that pharmacologic depletion of the antioxidant glutathione in mice prevents diet‐induced obesity, increases energy expenditure and locomotor activity, and enhances insulin sensitivity. These observations support a beneficial role of ROS in glucose homeostasis and warrant further research to define the regulation of metabolism and energy balance by ROS.

Transcriptional Regulation of S Phase Kinase-associated Protein 2 by NR4A Orphan Nuclear Receptor NOR1 in Vascular Smooth Muscle Cells

Background: The nuclear receptor NOR1 serves a mitogenic role; however, the mechanisms underlying this activity remain poorly understood. Results: NOR1 induces Skp2 transcription, leading to a decrease in p27 protein abundance. Conclusion: Skp2 constitutes a NOR1-regulated gene, which contributes to the mitogenic activity of this nuclear receptor. Significance: These studies detail a novel transcriptional cascade regulating proliferation. Members of the NR4A subgroup of the nuclear hormone receptor superfamily have emerged as key transcriptional regulators of proliferation and inflammation. NOR1 constitutes a ligand-independent transcription factor of this subgroup and induces cell proliferation; however, the transcriptional mechanisms underlying this mitogenic role remain to be defined. Here, we demonstrate that the F-box protein SKP2 (S phase kinase-associated protein 2), the substrate-specific receptor of the ubiquitin ligase responsible for the degradation of p27KIP1 through the proteasome pathway, constitutes a direct transcriptional target for NOR1. Mitogen-induced Skp2 expression is silenced in vascular smooth muscle cells (VSMC) isolated from Nor1-deficient mice or transfected with Nor1 siRNA. Conversely, adenovirus-mediated overexpression of NOR1 induces Skp2 expression in VSMC and decreases protein abundance of its target p27. Transient transfection experiments establish that NOR1 transactivates the Skp2 promoter through a nerve growth factor-induced clone B response element (NBRE). Electrophoretic mobility shift and chromatin immunoprecipitation assays further revealed that NOR1 is recruited to this NBRE site in the Skp2 promoter in response to mitogenic stimulation. In vivo Skp2 expression is increased during the proliferative response underlying neointima formation, and this transcriptional induction depends on the expression of NOR1. Finally, we demonstrate that overexpression of Skp2 rescues the proliferative arrest of Nor1-deficient VSMC. Collectively, these results characterize Skp2 as a novel NOR1-regulated target gene and detail a previously unrecognized transcriptional cascade regulating mitogen-induced VSMC proliferation.

The ApoB/ApoA1 Ratio is Associated with Metabolic Syndrome and its Components in a Chinese Population

In this study, we assessed whether the apolipoprotein B/apolipoprotein A1 ratio (ApoB/ApoA1) is related to metabolic syndrome (MS) and its components in an urban Chinese population. A total of 709 community residents were enrolled. Metabolic syndrome was defined according to the International Diabetes Federation definition in 2005. The high ApoB/ApoA1 group was defined as the gender-specific upper quartile of the ApoB/ApoA1 ratio. Insulin resistance (IR) was defined as the upper quartile of Homa-IR. The ApoB/ApoA1 ratio was significantly higher in subjects with MS, compared to those without (p < 0.05). After adjusting for age and gender, subjects with MS (odds ratio [OR] = 3.5) or IR (OR = 2.3) were more likely to be in the high ApoB/ApoA1 group. The ApoB/ApoA1 ratio increased significantly as the number of MS components increased (p < 0.05). Taken together, these data demonstrate that the ApoB/ApoA1 ratio is strongly associated with MS and its components in an urban Chinese population.

Serum complement C3 has a stronger association with insulin resistance than high-sensitivity C-reactive protein in women with polycystic ovary syndrome.

OBJECTIVE To compare the association of complement C3 (C3) versus high-sensitivity C-reactive protein (hs-CRP) with insulin resistance (IR) in women with polycystic ovary syndrome (PCOS). DESIGN Cross-sectional analysis. SETTING Clinical research center in China. PATIENT(S) One hundred thirty-three women with PCOS and 116 healthy, age-matched controls were recruited. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) One hundred one women with PCOS and 20 controls underwent hyperinsulinemic-euglycemic clamp to assess their insulin sensitivity, which was expressed as an M value. RESULT(S) Compared with controls, women with PCOS had a lower M value and higher C3 (1.37 ± 0. 34 vs. 1.10 ± 0.22 g/L) and hs-CRP levels (1.46 ± 2.29 vs. 0.49 ± 0.88 mg/L). In women with PCOS, C3 and hs-CRP negatively correlated with M value (r = -0.61 and r = -0.47, respectively). By regression analysis, C3 was found to have a greater impact on the M value (standardized coefficient β = -0.24) than did hs-CRP (standardized coefficient β = -0.13). After adjusting for body mass index (BMI), women with PCOS in the upper quartile were 4.30 times more likely to exhibit IR compared with those in the lower quartiles, whereas hs-CRP was not a statistically significant predictor of IR in women with PCOS. CONCLUSION(S) Compared with hs-CRP, serum C3 might be a stronger inflammatory marker of IR in women with PCOS.

Deficiency of the NR4A Orphan Nuclear Receptor NOR1 in Hematopoietic Stem Cells Accelerates Atherosclerosis

The NR4A orphan nuclear receptor NOR1 functions as a constitutively active transcription factor regulating cellular inflammation and proliferation. In this study, we used bone marrow transplantation to determine the selective contribution of NOR1 expression in hematopoietic stem cells to the development of atherosclerosis. Reconstitution of lethally irradiated apoE−/− mice with NOR1‐deficient hematopoietic stem cells accelerated atherosclerosis formation and macrophage recruitment following feeding a diet enriched in saturated fat. NOR1 deficiency in hematopoietic stem cells induced splenomegaly and monocytosis, specifically the abundance of inflammatory Ly6C+ monocytes. Bone marrow transplantation studies further confirmed that NOR1 suppresses the proliferation of macrophage and dendritic progenitor (MDP) cells. Expression analysis identified RUNX1, a critical regulator of hematopoietic stem cell expansion, as a target gene suppressed by NOR1 in MDP cells. Finally, in addition to inducing Ly6C+ monocytosis, NOR1 deletion increased the replicative rate of lesional macrophages and induced local foam cell formation within the atherosclerotic plaque. Collectively, our studies demonstrate that NOR1 deletion in hematopoietic stem cells accelerates atherosclerosis formation by promoting myelopoiesis in the stem cell compartment and by inducing local proatherogenic activities in the macrophage, including lesional macrophage proliferation and foam cell formation. Stem Cells 2014;32:2419–2429

Confirmatory Tests for the Diagnosis of Primary Aldosteronism: A Prospective Diagnostic Accuracy Study

The diagnosis of primary aldosteronism typically requires at least one confirmatory test. The fludrocortisone suppression test is generally accepted as a reliable confirmatory test, but it is cumbersome. Evidence from accuracy studies of the saline infusion test (SIT) and the captopril challenge test (CCT) has provided conflicting results. This prospective study aimed to evaluate the diagnostic accuracy of the SIT and CCT using fludrocortisone suppression test as the reference standard. One hundred thirty-five patients diagnosed with primary aldosteronism and 101 patients diagnosed with essential hypertension who completed the 3 confirmatory tests were included for the diagnostic accuracy analysis. The areas under the receiver–operator characteristics curves of the CCT and SIT were 0.96 (95% confidence interval [CI], 0.92–0.98) and 0.96 (95% CI, 0.92–0.98), respectively, using post-test plasma aldosterone concentration (PAC) for diagnosis. However, the areas under the receiver–operator characteristics curves of the CCT decreased to 0.71 (95% CI, 0.65–0.77) when the PAC suppression percentage was used to diagnose primary aldosteronism. The optimal cutoff of PAC post-CCT was set at 11 ng/dL, resulting in a sensitivity of 0.90 (95% CI, 0.84–0.95) and a specificity of 0.90 (95% CI, 0.83–0.95), which were not significantly different from those of SIT (with PAC post-SIT set at 8 ng/dL, sensitivity: 0.85 [95% CI, 0.78–0.91], P=0.192; specificity: 0.92 [95% CI, 0.85–0.97], P=0.551). In conclusion, both CCT and SIT are accurate alternatives to the more complex fludrocortisone suppression test. Because CCT is safe and much easier to perform, it may serve as a more feasible alternative. When interpreting the results of CCT, PAC post-CCT is highly recommended.

Telomerase Reverse Transcriptase Deficiency Prevents Neointima Formation Through Chromatin Silencing of E2F1 Target Genes.

Objective— Aberrant proliferation of smooth muscle cells (SMC) in response to injury induces pathological vascular remodeling during atherosclerosis and neointima formation. Telomerase is rate limiting for tissue renewal and cell replication; however, the physiological role of telomerase in vascular diseases remains to be determined. The goal of the present study was to determine whether telomerase reverse transcriptase (TERT) affects proliferative vascular remodeling and to define the molecular mechanism by which TERT supports SMC proliferation. Approach and Results— We first demonstrate high levels of TERT expression in replicating SMC of atherosclerotic and neointimal lesions. Using a model of guidewire-induced arterial injury, we demonstrate decreased neointima formation in TERT-deficient mice. Studies in SMC isolated from TERT-deficient and TERT overexpressing mice with normal telomere length established that TERT is necessary and sufficient for cell proliferation. TERT deficiency did not induce a senescent phenotype but resulted in G1 arrest albeit hyperphosphorylation of the retinoblastoma protein. This proliferative arrest was associated with stable silencing of the E2F1-dependent S-phase gene expression program and not reversed by ectopic overexpression of E2F1. Finally, chromatin immunoprecipitation and accessibility assays revealed that TERT is recruited to E2F1 target sites and promotes chromatin accessibility for E2F1 by facilitating the acquisition of permissive histone modifications. Conclusions— These data indicate a previously unrecognized role for TERT in neointima formation through epigenetic regulation of proliferative gene expression in SMC.