Zhikun Guo

Endothelin-1 upregulation mediates aging-related cardiac fibrosis

Endothelin-1 (ET-1) plays a major role in regulating myocardial fibrosis in several pathological conditions, such as hypertension and diabetes. Aging is an independent risk factor for myocardial fibrosis. We hypothesized that ET-1 upregulation may be a basis of enhanced collagen synthesis in the senescent fibroblasts resulting in cardiac fibrosis with aging. To examine this hypothesis, we cultured mouse cardiac fibroblasts to passage-30 (P30). β-Galactosidase activity and several other aging markers were markedly increased in P30 (vs. P3) fibroblasts, indicating that these cells were indeed undergoing senescence. Importantly, ET-1 expression was markedly upregulated in P30 (vs. P3) fibroblasts. Of note, estrogen receptor-α (ER-α), an important negative regulator of ET-1, was downregulated in P30 fibroblasts. We also studied aged (130-weeks old, female) mice hearts, and observed that ET-1 was upregulated and ER-α was downregulated in these hearts (vs. 6-week old mice hearts, female). Similar observations were made in the fibroblasts isolated from aged mice hearts. ET-1 upregulation with aging was also seen in ≈70-year old (vs. ≈30-year old) human heart sections. In concert with ET-1 upregulation, the expression of fibronectin and collagens was found to be markedly increased in P30 cardiac fibroblasts in culture, fibroblasts isolated from the aged mice hearts, and in aged human hearts. Interestingly, inhibition of ET-1 in the senescent P30 fibroblasts by 2 different strategies (the use of siRNA and the use of endothelin converting enzyme inhibitors) markedly suppressed expression of fibrosis signals. Further, treatment with synthetic ET-1 enhanced fibronectin and collagen expression in P3 cardiac fibroblasts. These observations in mice and human hearts suggest that aging-related cardiac fibrosis is, at least partially, dependent on the upregulation of ET-1.

Effects of linagliptin and liraglutide on glucose- and angiotensin II-induced collagen formation and cytoskeleton degradation in cardiac fibroblasts in vitro.

Aim:Glucagon-like peptide-1 (GLP-1) agonists and dipeptidyl peptidase-4 (DPP-4) inhibitors can not only lower blood glucose levels, but also alleviate cardiac remodeling after myocardial ischemia and hypertension. In the present study, we investigated the effects of a DPP-4 inhibitor (linagliptin) and a GLP-1 activator (liraglutide) on glucose- and angiotensin II (Ang II)-induced collagen formation and cytoskeleton reorganization in cardiac fibroblasts in vitro, and elucidated the related mechanisms.Methods:Cardiac fibroblasts were isolated from the hearts of 6-week-old C57BL/6 mice, and then exposed to different concentrations of glucose or Ang II for 24 h. The expression of fibrotic signals (fibronectin, collagen-1, -3 and -4), as well as ERK1/2 and NF-κB-p65 in the fibroblasts was examined using Western blotting assays. F-actin degradation was detected under inverted laser confocal microscope in fibroblasts stained with Rhodamine phalloidin.Results:Glucose (1–40 mmol/L) and Ang II (10−8–10−5 mol/L) dose-dependently increased the expression of fibronectin, collagens, phospho-ERK1/2 and phospho-NF-κB-p65 in cardiac fibroblasts. High concentrations of glucose (≥40 mmol/L) and Ang II (≥10−6 mol/L) caused a significant degradation of F-actin (less assembly F-actin fibers and more disassembly fibers). ERK1/2 inhibitor U0126 (10 μmol/L) and NF-κB inhibitor JSH-23 (10 μmol/L) both markedly suppressed glucose- and angiotensin II-induced fibronectin and collagen expressions in cardiac fibroblasts. Furthermore, pretreatment with liraglutide (10–100 nmol/L) or linagliptin (3 and 30 nmol/L) significantly decreased glucose- and Ang II-induced expression of fibrotic signals, phospho-ERK1/2 and phospho-NF-κB-p65 in cardiac fibroblasts. Moreover, pretreatment with liraglutide (30 nmol/L) or liraglutide (100 nmol/L) markedly inhibited glucose-induced F-actin degradation, however, only liraglutide inhibited Ang II-induced F-actin degradation.Conclusion:Linagliptin and liraglutide inhibit glucose- and Ang II-induced collagen formation in cardiac fibroblasts via activation of the ERK/NF-κB/pathway. Linagliptin and liraglutide also markedly inhibit glucose-induced F-actin degradation in cardiac fibroblasts, but only liraglutide inhibits Ang II-induced F-actin degradation.

LOX-1 Deletion Limits Cardiac Angiogenesis in Mice Given Angiotensin II

Lectin-like oxidized low-density lipoprotein (ox-LDL) receptor-1 (LOX-1) is a major receptor for ox-LDL in endothelial cells. Its activation regulates endothelial proliferation, differentiation, migration and apoptosis. Recent in vitro studies show that LOX-1 activation by ox-LDL and angiotensin II (Ang II) induces angiogenesis via activation of NADPH oxidase and subsequent increase in ROS production. In this study, we investigated the effect of LOX-1 gene deletion (LOX-1 knockout or KO mice) on angiogenesis in response to prolonged Ang II infusion in vivo. Our studies showed that Ang II (vs. saline) infusion enhanced capillary formation in subcutaneously injected Matrigel® plugs. Ang II infusion also resulted in marked angiogenesis in the hearts as determined by CD31 immunopositivity. There was an increased expression (RT-PCR and Western blotting) of CD31 and VEGF in the hearts of mice infused with Ang II, indicating pro-angiogenic miliue. More importantly, LOX-1 KO mice reveled markedly limited angiogenesis in the Matrigel® plugs as well as in the hearts despite similar infusion with Ang II (all P < 0.05 vs. wild-type mice). In addition, the hearts of LOX-1 KO mice had attenuated expression of pro-inflammatory and angiogenic signals MCP-1 and IL-1β following Ang II Infusion. Lastly, the rise in blood pressure in response to Ang II was less in the LOX-1 KO mice (P < 0.05 vs. wild-type mice). Our findings suggest that LOX-1 participates in angiogenesis in hypertension, which may be related to a state of inflammation.

Overexpression of CyclinA2 ameliorates hypoxia‑impaired proliferation of cardiomyocytes

Hypoxia is a primary mediator for cell survival, and has been reported to inhibit cardiomyocyte proliferation in fetal and neonatal hearts. CyclinA2 is a key regulator of cell proliferation. Whether CyclinA2 affects cardiomyocyte proliferation in hypoxic conditions remains unexamined. This study was designed to investigate the roles of CyclinA2 expression on hypoxia-impaired cardiomyocyte proliferation. Cardiomyocytes were isolated from neonatal rats and randomly separated into six groups: Control, hypoxia, enhanced green fluorescent protein (EGFP)-Adv, EGFP-Ccna2, EGFP-Adv + hypoxia and EGFP-Ccna2 + hypoxia. The cells in the control group were cultured in a general cell incubator; the cells in the hypoxia group were placed in a hypoxic chamber for 12 h; the cells in the EGFP-Adv and EGFP-Ccna2 groups were separately transfected with EGFP-adenovirus capsids or EGFP-adenovirus capsids with CyclinA2 cDNA for 18 h, and then placed in a general incubator for an additional 12 h; the cells in the EGFP-Adv + hypoxia and EGFP-Ccna2 + hypoxia groups were separately transfected with EGFP-adenovirus capsids or EGFP-adenovirus capsids with CyclinA2 cDNA for 18 h, and then placed in a hypoxia chamber for an additional 12 h. CyclinA2 expression was measured using immunochemical staining and western blot analysis, and cardiomyocyte proliferation was measured using the cell counting kit 8. GFP fluorescence indicated a high transfection efficiency (>80%), and immunochemical staining showed that CyclinA2 was mainly distributed in the nucleus. CyclinA2 expression was downregulated following exposure to hypoxia for 12 h. Cardiomyocyte proliferation was also significantly decreased following exposure to hypoxia for 12 h. However, compared with the EGFP-Adv group, CyclinA2 expression and cardiomyocyte proliferation was markedly increased in the EGFP-Ccna2 group. Furthermore, compared with the EGFP-Adv + hypoxia group, CyclinA2 expression and cell proliferation were markedly increased in the EGFP-Ccna2 + hypoxia group. These findings indicate that CyclinA2 upregulation improves cardiomyocyte proliferation in hypoxic conditions.

Inflammation, Autophagy, and Apoptosis After Myocardial Infarction

Background There is evidence for inflammation, autophagy, and apoptosis in the ischemic heart. Autophagy is a physiologic process for tissue survival. Apoptosis, on the other hand, is a mechanism that serves to clear the debris in the setting of tissue injury. The balance between autophagy and apoptosis may be important in cell survival and cardiac function. Methods and Results We examined the interplay of inflammation and myocyte autophagy and apoptosis during the ischemic process. We subjected mice to total left coronary artery ligation and studied these animals for up to 4 weeks. The inflammatory (tumor necrosis factor [TNF]‐α, monocyte chemoattractant protein‐1, interleukin‐6, and interleukin‐1β) and autophagic signals (light chain‐3 and beclin‐1) were strongest during the first week and then began to decline. However, the apoptotic signals peaked at week 2 after left coronary artery ligation, and the elevated levels persisted until the end of the fourth week. To elucidate the role of inflammation in the regulation of myocyte autophagy and apoptosis, we administered TNF‐α inhibitor (CAS1049741‐03‐8, Millipore, Burlington, MA) to the mice daily during the first week of myocardial infarction. Anti‐TNF‐α therapy reduced the levels of inflammatory cytokines and the inflammatory cell infiltration in and around the infarct area. However, cardiac function measured by echocardiography (fractional shortening and ejection fraction) worsened with anti‐TNF‐α therapy. More importantly, application of TNF‐α inhibitor markedly inhibited autophagy and promoted myocyte apoptosis in the border zone. Conclusions These observations suggest that inflammatory response may be protective in the early stage of the myocardial infarction through stimulation of myocyte autophagy. Anti‐inflammatory treatment early after coronary occlusion may have an adverse effect.

Internal associations and dynamic expression of c-kit and nanog genes in ventricular remodelling induced by adriamycin

The present study aimed to investigate the dynamic expression of the c-kit and nanog genes in rats with left ventricular remodelling induced by adriamycin (ADR), and explore its internal association and mechanism of action. Sprague-Dawley male rats were randomly divided into a normal control group and a heart failure model group. Heart failure was induced by a single intraperitoneal injection of ADR (4 mg/kg) weekly for six weeks. The normal control group was given the same amount of saline. At the eighth week, rat cardiac function was examined to demonstrate the formation of heart failure. The rat hearts were harvested frozen and sectioned, and the expression levels of the nanog and c-kit genes in the myocardial tissue samples were detected using immunohistochemistry, immunofluorescence and reverse transcription-polymerase chain reaction (RT-PCR). Hematoxylin and eosin staining demonstrated various pathological changes in the myocardial cells in the heart failure model group, whereas myocardial infarction was not observed in the normal control group. Immunohistochemistry and immunofluorescence demonstrated that nanog-positive cells were predominantly expressed in the vascular endothelium, with a few myocardial cells and stem cells in normal myocardium. The expression levels of c-kit and nanog in the myocardium of the rats with heart failure decreased significantly. c-kit-positive cells clustered together in the epicardium and its vicinity, and c-kit expression significantly decreased in the myocardium of rats with heart failure, as compared with normal rats. In both groups, some cells co-expressed both the c-kit and nanog genes. The RT-PCR results demonstrated that the expression levels of the two genes in the heart failure model group were significantly lower compared with those in the normal control group (P<0.05). In conclusion, the c-kit- and nanog-positive stem cells decreased in the myocardium of the rats with left ventricular remodelling induced by ADR. Their abnormal expression was significantly correlated with left ventricular remodelling, thereby indicating an internal association (influences of two indexes in the experimental group and control group) between them.

Functions of MicroRNAs in Angiogenesis

Angiogenesis is defined as formation and growth of new blood vessels that sprout from existing vascular network. Angiogenesis plays a very important role in the physiological and pathological situations such as development, ischemia, atherosclerosis, wound healing, and cancer growth and metastasis. MicroRNAs (miRNAs or miRs) are endogenous, short, noncoding RNAs found in eukaryotic cells. MiRs are major posttranscriptional regulators that negatively regulate gene expression by binding to their target messenger RNAs for degradation and/or translational repression. The main function of miRs is gene regulation. MiRs have been found to modulate many pathophysiological process including cell differentiation, contraction, migration, proliferation, apoptosis, and tissue inflammation. There are more than 1, 000 miRs in human genome, some of them are involved in angiogenesis. In this review, we will summarize the recent progress on function of miRs in angiogenesis.

Expression of N-cadherin proteins in myocardial hypertrophy in rats

The aim of the present study was to examine the expression of N-cadherin in the myocardial tissues of isoproterenol-induced myocardial hypertrophy in rats. In addition, the present study provided morphological data to investigate the signal transduction mechanisms of myocardial hypertrophy and reverse myocardial hypertrophy. A myocardial hypertrophy model was established by subcutaneously injecting isoprenaline into healthy adult Sprague-Dawley rats. The myocardial tissue was collected, embedded in conventional paraffin, sectioned and stained with hematoxylin and the pathological changes were observed. The expression and distribution of N-cadherin were detected by immunohistochemistry (IHC) and the changes in mRNA expression of N-cadherin in the myocardial tissues of rats were detected by reverse transcription polymerase chain reaction. Image analysis software was used to quantitatively analyze the expression of N-cadherin. The IHC and immunofluorescence results showed that there was no statistically significant difference between the experimental and control groups in the positive expression of N-cadherin. Furthermore, mRNA expression of N-cadherin, in the myocardial tissues of rats, was consistent with the IHC and immunofluorescence results. Thus, N-cadherin may have a significant function in the occurrence and development of myocardial hypertrophy.