Regulatory crosstalk between KLF5, miR-29a and Fbw7/CDC4 cooperatively promotes atherosclerotic development.

Atherogenesis is a chronic inflammatory process that involves complex interactions between endothelial dysfunction, lipid deposition and vascular smooth-muscle cell (VSMC) proliferation. However, the molecular mechanism is still unclear. We found that a pro-atherosclerotic factor (oxLDL) induced the expression of Krüppel-like factor 5 (KLF5), which in turn increased miR-29a expression levels. The increased miR-29a was retained within HASMCs and down-regulated Fbw7/CDC4 expression by targeting the 3´UTR of Fbw7/CDC4, subsequently increasing KLF5 stability by reducing the Fbw7/CDC4-dependent ubiquitination of KLF5, forming a positive feedback loop to enhance VSMC proliferation and promote atherogenesis. These results indicate a potentially important role for the oxLDL-activated feedback mechanism in VSMC proliferation and atherogenesis. Suppression of miR-29a may be an effective way to attenuate atherosclerosis. In conclusion, our data are the first to reveal that the regulatory crosstalk between KLF5, miR-29a, and Fbw7/CDC4 cooperatively promotes atherosclerotic development.

SUMOylation of KLF4 acts as a switch in transcriptional programs that control VSMC proliferation.

The regulation of vascular smooth muscle cell (VSMC) proliferation is an important issue due to its major implications for the prevention of pathological vascular conditions. The objective of this work was to assess the function of small ubiquitin-like modifier (SUMO)ylated Krϋppel-like transcription factor 4 (KLF4) in the regulation of VSMC proliferation in cultured cells and in animal models with balloon injury. We found that under basal conditions, binding of non-SUMOylated KLF4 to p300 activated p21 (p21(WAF1/CIP1))transcription, leading to VSMC growth arrest. PDGF-BB promoted the interaction between Ubc9 and KLF4 and the SUMOylation of KLF4, which in turn recruited transcriptional corepressors to the p21 promoter. The reduction in p21 enhanced VSMC proliferation. Additionally, the SUMOylated KLF4 did not affect the expression of KLF4, thereby forming a positive feedback loop enhancing cell proliferation. These results demonstrated that SUMOylated KLF4 plays an important role in cell proliferation by reversing the transactivation action of KLF4 on p21 induced with PDGF-BB.

miR-200c-SUMOylated KLF4 feedback loop acts as a switch in transcriptional programs that control VSMC proliferation.

The regulation of vascular smooth muscle cell (VSMC) proliferation is an important issue because it has major implications for the prevention of pathological vascular conditions. Using microRNA array screen, we found the expression levels of 200 unique miRNAs in hyperplasic tissues. Among them, miR-200c expression substantially was down-regulated. The objective of this work was to assess the function of miR-200c and SUMOylated Krϋppel-like transcription factor 4 (KLF4) in the regulation of VSMC proliferation in both cultured cells and animal models of balloon injury. Under basal conditions, we found that miR-200c inhibited the expression of KLF4 and the SUMO-conjugating enzyme Ubc9. Upon PDGF-BB treatment, Ubc9 interacted with and promoted the SUMOylation of KLF4, which allowed the recruitment of transcriptional corepressors (e.g., nuclear receptor corepressor (NCoR) and HDAC2) to the miR-200c promoter. The reduction in miR-200c levels led to increased target gene expression (e.g., Ubc9 and KLF4), which further repressed miR-200c levels and accelerated VSMC proliferation. These results demonstrate that induction of a miR-200c-SUMOylated KLF4 feedback loop is a significant aspect of the PDGF-BB proliferative response in VSMCs and that targeting Ubc9 represents a novel approach for the prevention of restenosis.

Tongxinluo (TXL), a Traditional Chinese Medicinal Compound, Improves Endothelial Function After Chronic Hypoxia Both In Vivo and In Vitro.

Vascular injury after chronic hypoxia leads to endothelial injury and structural damage to tight junctions (TJs), thereby resulting in a variety of cardiovascular diseases. Thus, attenuating hypoxia-induced damage has great significance for the prevention and treatment of cardiovascular disease. The aim of this study was to investigate whether the endothelial protection conferred by tongxinluo (TXL), a traditional Chinese medicinal compound, is related to its regulation of TJ protein expression. In vivo, we found that TXL could promote hypoxia-induced angiogenesis in lung and liver tissue. In vitro, we found that CoCl2 treatment significantly reduced the expression of the TJ proteins occludin, claudin-1, VE-cadherin, and beta-catenin in cultured human cardiac microvascular endothelial cells. TXL pretreatment abrogated the CoCl2-induced downregulation of these TJ proteins. Conversely, overexpression of Krüppel-like factor 4 (KLF4) inhibited the expression of TJ proteins in human cardiac microvascular endothelial cells, an effect that was reversed by TXL pretreatment. Further experiments showed that TXL could promote endothelial cell proliferation by increasing KLF4 phosphorylation, thereby reversing the effect of KLF4 on the expression of TJ proteins. These findings provide a new molecular mechanism for the TXL-induced increase in TJ protein expression.

Chinese medicine Tongxinluo increases tight junction protein levels by inducing KLF5 expression in microvascular endothelial cells.

Tongxinluo (TXL) is a compound prescription formulated according to the meridian theory of traditional Chinese medicine. It may play an important role in cardiovascular protection by improving endothelial cell function. The aim of present study was to investigate whether endothelial protection with TXL is related to its regulation of tight junction protein expression. Human cardiac microvascular endothelial cells (HCMECs) were cultured and treated with 10(-7) mol l(-1) angiotensin II (Ang II) and the different doses of TXL; the expression of tight junction proteins occludin, claudin, VE-cadherin and beta-catenin was determined by Western blotting and real-time PCR. Gain-of-function and loss-of-function of Krüppel-like factor 5 (KLF5) were carried out in HCMEC transfected with either KLF5 adenovirus pAd-KLF5 or siRNA specific for KLF5. Angiotensinogen transgenic mice were treated with TXL by oral administration of TXL of 0.75 g kg(-1) day(-1) , and immunohistochemical staining was performed with antioccludin, anticlaudin, anti-VE-cadherin, antibeta-catenin and anti-KLF5 antibodies. Ang II treatment significantly reduced the expression of tight junction proteins occludin, claudin, VE-cadherin and beta-catenin in cultured HCMECs. TXL pretreatment could abrogate the down-regulation of these tight junction proteins induced by Ang II. Ang II treatment also decreased KLF5 expression at the mRNA and protein levels; TXL pretreatment markedly reversed the inhibitory effect of Ang II on KLF5 expression. Gain-of-function and loss-of-function of KLF5 showed that KLF5 mediated the expression of tight junction proteins in HCMECs. TXL-enhanced expression of the tight junction proteins was mediated by KLF5. In angiotensinogen transgenic mice, TXL also increased the tight junction protein levels by inducing KLF5 expression. Chinese medicine TXL increases tight junction protein levels by inducing KLF5 expression in microvascular endothelial cells.

miR-29a regulates vascular neointimal hyperplasia by targeting YY1.

OBJECTIVES: The formation of vascular neointima is mainly related to impairment of the vascular endothelial barrier and abnormal proliferation and migration of smooth muscle cells. The objective of this study was to investigate whether miR-29a exerts any promoting effect on the vascular neointimal hyperplasia and if so, its mechanism. MATERIALS AND METHODS: RT-qPCR was performed to determine expression of miR-29a in vascular smooth muscle cells (VSMC) and vascular neointimal hyperplasia. To further understand its role, we restored its expression in VSMCs by transfection with miR-29a mimics or inhibitors. Effects of miR-29a on cell proliferation were also determined. RESULTS: In this study, we used two kinds of model to observe the role of miR-29a in neointimal hyperplasia induced by carotid ligation or balloon injury. The major findings were that: (i) miR-29a overexpression promoted neointimal hyperplasia induced by carotid ligation; (ii) miR-29a increased proliferation of VSMCs, one aspect of which was by targeting expression of Ying and yang 1 protein (YY1), a negative regulator of Cyclin D1. A further aspect, was by increasing expression of Krüppel-like factor 5, a positive regulator of Cyclin D1, thereby allowing formation a synergistic effect. (iii) Tongxinluo (TXL), a traditional Chinese medicine reduced neointimal formation in ligated vessels by inhibiting VSMC proliferation and migration. CONCLUSIONS: These findings provide a new molecular mechanism of TXL in decreasing neointima hyperplasia.

Disseminated cryptococcosis caused by Cryptococcus neoformans a case report and review / 中国热带医学

@#Abstract: To analyze the clinical, therapeutic and laboratory characteristics of disseminated cryptococcosis caused by Cryptococcus neoformans invading the blood stream in patient with liver cirrhosis and splenectomy. A 30-year-old male underwent splenectomy plus pericardial devascularization due to "splenomegaly and hypersplenism" in March in 2016. The patient had intermittent fever after operation for many times, and successively accompanied with back pain, left lower limb abscess and right hip pain. The highest body temperature was 39 ℃. CT and MRI revealed the lung lesion and multiple bone destruction. During that period, the effect of antibiotics was not good. On April 19th, 2017, Gram's stain, India ink stain, API 32C, Vitek 2 Compact, ribosomal ITS and IGS sequence analysis were performed to identify the strain isolated from the pus and blood stream. The serum of the patient was detected for cryptococcal antigen. Antifungal susceptibility test was used to determine drug sensitivity and minimum inhibitory concentration (MIC). The Cryptococcus neoformans isolated from fresh pus specimen showed a prominent, thick capsule after India ink stain. The colonies isolated from pus and blood stream were identified Cryptococcus neoformans using API 32C, Vitek 2 Compact, and sequence analysis of rDNA ITS and IGS. Cryptococcal capsule antigen was positive. The minimal inhibitory concentrations of 5-Flucytosine, amphotericin B, fluconazole, itriconazole, voriconazole against the isolate were <4 μg/mL, <0.5 μg/mL, 4 μg/mL, ≤0.25 μg/mL, 0.125 μg/mL respectively. The patient was initially treated with intravenous amphotericin B and flucytosine. After anti-Cryptococcus treatment for two months, the patient clinically improved, and the lesions were reduced on a follow-up CT scan. The patient made a full functional recovery after treatment for six months. Cryptococcosis has hidden onset, atypical clinical symptoms and lack of specificity. Blood stream is the main channel for Cryptococcus to spread and involve many organs of the whole body, including skin, bone and so on. Therefore, early use of blood culture to monitor blood flow dissemination, actively removing the primary focus and cutting off the infection route in time and carrying out effective anti-Cryptococcus treatment are conducive to the patient's early recovery.