UFM1 inhibits hypoxia-induced angiogenesis via promoting proteasome degradation of HIF-1α.

Angiogenesis is crucial for blood flow recovery and ischemic tissue repair of peripheral artery disease (PAD). Exploration of new mechanisms underlying angiogenesis will shed light on the treatment of PAD. Ubiquitin-fold modifier 1 (UFM1), a newly identified ubiquitin-like molecule, has been discovered to be involved in various pathophysiological processes. However, the role of UFM1 in the pathogenesis of PAD, especially in endothelial angiogenesis remains obscure, and we aimed to clarify this issue in this study. We initially found UFM1 was significantly upregulated in gastrocnemius muscles of PAD patients and hind limb ischemia mice. And UFM1 was mainly colocalized with endothelial cells in ischemic muscle tissues. Further, elevated expression of UFM1 was observed in hypoxic endothelial cells. Subsequent genetic inhibition of UFM1 dramatically enhanced migration, invasion, adhesion, and tube formation of endothelial cells under hypoxia. Mechanistically, UFM1 reduced the stability of hypoxia-inducible factor-1α (HIF-1α) and promoted the von Hippel-Lindau-mediated K48-linked ubiquitin-proteasome degradation of HIF-1α, which in turn decreased angiogenic factor VEGFA expression and suppressed VEGFA related signaling pathway. Consistently, overexpression of UFM1 inhibited the angiogenesis of endothelial cells under hypoxic conditions, whereas overexpression of HIF-1α reversed this effect. Collectively, our data reveal that UFM1 inhibits the angiogenesis of endothelial cells under hypoxia through promoting ubiquitin-proteasome degradation of HIF-1α, suggesting UFM1 might serve as a potential therapeutic target for PAD.

PS-341 alleviates chronic low-grade inflammation and improves insulin sensitivity through the inhibition of TM4 (UBAC2) degradation.

BACKGROUND: The TM4 (UBAC2) protein, which contains 4 transmembrane domains and one ubiquitin binding domain, is mainly expressed in cell and nuclear membranes. The current research aimed to explore the role of TM4 in metabolic inflammation and to examine whether the ubiquitin-proteasome inhibitor PS-341 could regulate the function of TM4. METHODS: The metabolic phenotypes of TM4 knockout (KO) mice were studied. We next explored the association between the polymorphisms of TM4 and obesity in a Chinese Han population. TM4 expression in the visceral fat of obese patients who underwent laparoscopic cholecystectomy was also analysed. Finally, the effect of PS-341 on the degradation and function of the TM4 protein was investigated in vivo and in vitro. RESULTS: TM4 KO mice developed obesity, hepatosteatosis, hypertension, and glucose intolerance under a high-fat diet. TM4 counterregulated Nur77, IKKß, and NF-kB both in vivo and in vitro. The TM4 SNP rs147851454 is significantly associated with obesity after adjusting for age and sex (OR 1.606, 95% CI 1.065-2.422 P = 0.023) in 3394 non-diabetic and 1862 type 2 diabetic adults of Han Chinese. TM4 was significantly downregulated in the visceral fat of obese patients. PS-341 induced TM4 expression through inhibition of TM4 degradation in vitro. In db/db mice, PS-341 administration led to downregulation of Nur77/IKKß/NF-κB expression in visceral fat and liver, and alleviation of hyperglycaemia, hypertension, and glucose intolerance. The hyperinsulinaemic-euglycaemic clamp demonstrated that PS-341 improved the glucose infusion rate and alleviated insulin resistance in db/db mice. CONCLUSIONS: PS-341 alleviates chronic low-grade inflammation and improves insulin sensitivity through inhibition of TM4 degradation.

Knockdown of Tubulin Polymerization Promoting Protein Family Member 3 inhibits cell proliferation and invasion in human colorectal cancer.

Tubulin Polymerization Promoting Protein Family Member 3 (TPPP3), a member of the TPPP protein family, has been reported to play important roles in initiation and progression of human cancers. However, the expression and underlying function of TPPP3 in colorectal cancer (CRC) have not yet been fully clarified. In this study, the mRNA and protein levels of TPPP3 in 96 clinical CRC specimens were determined by RT-PCR and immunohistochemistry. The relation between TPPP3 expression and clinicopathologic characteristics and overall survival (OS) were evaluated. Further experiments showed that knockdown of TPPP3 inhibited cell proliferation, migration and invasion and induced cell apoptosis in vitro. In addition, TPPP3 silencing resulted in a decrease of angiogenesis and S phase fraction. Thus, our results suggested that TPPP3 played an important role in CRC progress and might serve as novel therapeutic target for CRC treatment.

Knockdown of Tubulin Polymerization Promoting Protein Family Member 3 Suppresses Proliferation and Induces Apoptosis in Non-Small-Cell Lung Cancer.

Our previous studies demonstrated that depletion of tubulin polymerization promoting protein family member 3 (TPPP3) inhibits proliferation and induces apoptosis of HeLa cells. However, the expression and roles of TPPP3 in cancers remain largely unknown. In this study, we investigated the expression of TPPP3 in clinicopathological correlations in non-small-cell lung cancer (NSCLC) samples by immunohistochemistry. TPPP3 expression was significantly upregulated in NSCLC tissues, and high TPPP3 expression was positively associated with tumor size, lymph node metastasis, clinical stage, and poor survival. Furthermore, knockdown of TPPP3 by shRNA significantly inhibited cell proliferation and induced cell apoptosis and cell cycle arrest in vitro. In addition, depletion of TPPP3 inhibited lung cancer growth in vivo in the xenografts of H1299 cells; this effect was accompanied by the suppression of Ki67 expression. Our data suggested that TPPP3 might act as an oncogene in NSCLC. TPPP3 warrants consideration as a therapeutic candidate with anti-tumor potential.

Cross-sectional analysis of serum calcium levels for associations with left ventricular hypertrophy in normocalcemia individuals with type 2 diabetes.

BACKGROUND: Left ventricular hypertrophy (LVH) is prevalent in patients with type 2 diabetes mellitus (T2DM). Recent studies show that an increase in albumin-adjusted serum calcium level is associated with an elevated risk of T2DM. We speculate that increased serum calcium levels in T2DM patients are related to LVH prevalence. METHODS: In this echocardiographic study, 833 normocalcemia and normophosphatemia patients with T2DM were enrolled. The associations between serum calcium and metabolic parameters, left ventricular mass index (LVMI), as well as the rate of LVH were examined using bivariate linear correlation, multivariate linear regression and logistic regression, respectively. The predictive performance of serum calcium for LVH was evaluated using the area under the receiver operating characteristic curve (AUC). RESULTS: Patients with LVH have significantly higher serum calcium than those without LVH. Serum calcium was positively associated with total cholesterol, triglycerides, low-density lipoprotein cholesterol, serum uric acid, HOMA-IR and fasting plasma glucose. Multivariate linear regression analysis demonstrated that serum calcium was independently associated with LVMI (p < 0.001). In comparison with patients in the lowest serum calcium quartile, the odds ratio (OR) for LVH in patients in the highest quartile was 2.909 (95% CI 1.792-4.720; p < 0.001). When serum calcium was analyzed as a continuous variable, per 1 mg/dl increase, the OR (95% CI) for LVH was [2.400 (1.552-3.713); p < 0.001]. Serum calcium can predict LVH (AUC = 0.617; 95% CI (0.577-0.656); p < 0.001). CONCLUSIONS: Albumin-adjusted serum calcium is associated with an increased risk of LVH in patients with T2DM.

Albuminuria is associated with left ventricular hypertrophy in patients with early diabetic kidney disease.

Aims. Left ventricular hypertrophy (LVH) and albuminuria are both markers for cardiovascular diseases (CVDs) in patients with type 2 diabetes mellitus (T2DM). We speculate that albuminuria in T2DM patients with early diabetic kidney disease (DKD) could predict LVH. Methods. 333 diabetic patients (219 non-DKD and 114 early DKD) were enrolled. The association between albuminuria and LVMI was examined using multivariate linear regression and logistic regression. Results. The rate of LVH was significantly higher in patients with early DKD versus those without DKD (57.0% versus 32.9%; P < 0.001). Multivariate linear regression analysis demonstrated that albuminuria status (no, micro-, and macroalbuminuria; P < 0.001), age (P < 0.001), systolic blood pressure (P = 0.0578), and the use of ACEI/ARB drug (P < 0.001) were independently associated with LVMI. The risks were substantially higher for LVH in the microalbuminuria group (odds ratio 2.473 (95% confidence interval 1.370-4.464)) and macroalbuminuria group (odds ratio 3.940 (95% confidence interval 1.553-9.993)) compared with that in non-DKD group. Concentric hypertrophy was the most common geometric pattern in patients with early DKD (36.0%), followed by eccentric hypertrophy (21.0%). Conclusions. Albuminuria is associated with higher LVMI and higher rate of LVH in patients with early phase DKD.

Association between Brachial-Ankle pulse wave velocity and cardiac autonomic neuropathy in type 2 diabetes.

BACKGROUND: Cardiac autonomic neuropathy (CAN) is a common complication of type 2 diabetes mellitus (T2DM). Brachial-ankle pulse wave velocity (baPWV) is known to be a good surrogate marker of vascular damages. The goal of this study was to investigate the relationship between BaPWV and CAN in T2DM. METHODS: A total of 148 patients who had no apparent history of cardiovascular condition were enrolled consecutively in this study. The correlation between increased baPWV and CAN was analyzed. CAN was evaluated by five standard cardiovascular reflex tests (CARTs) according to the Ewing's protocol: 1) heart rate variation during deep breathing, 2) heart rate response to standing, 3) Valsalva maneuver, 4) postural systolic blood pressure (BP) change, 5) Sustained handgrip test. CAN was defined as the presence of at least two abnormal tests. RESULTS: The mean age of patients was 59.8 ± 7.8 years. The mean duration of diabetes was 6.0(2.0-11.0) years. The mean baPWV was 1665.5(1482.0-1940.0) cm/sec. Subjects with CAN were older and had high BMI, baPWV compared with those without CAN. The proportion of patients with diabetic peripheral neuropathy was higher in subjects with CAN. After adjusting for other confounding risk factors, baPWV (odds ratio = 8.496, 95% CI: 1.216-59.348; P = 0.031) remained as independent risk factors for CAN. The number of abnormal CARTs increased gradually with increasing baPWV (correlation coefficient =0.255, p = 0.002). CONCLUSION: Increased baPWV was significantly correlated with CAN in patients with type 2 diabetes.

Association of lipid profiles and the ratios with arterial stiffness in middle-aged and elderly Chinese.

BACKGROUND: Serum lipids and the ratios are known to be associated with the cardiovascular diseases (CVD). However, the associations of serum lipids and the ratios related to arterial stiffness are unclear. We sought to compare the strength of these serum lipids and the ratios with arterial stiffness assessing by brachial-ankle pulse wave velocity (baPWV) in the middle-aged and elderly Chinese subjects. METHODS: A total number of 1133 Chinese aged from 50 to 90 years old were recruited from Shanghai downtown district. The serum lipids, baPWV and major cardiovascular risk factors of the participants were measured. RESULTS: Participants with high baPWV exhibited higher levels of non-HDL-c, TC/HDL-c, TG/HDL-c, LDL-c/HDL-c, and non-HDL-c/HDL-c, while HDL-c worked in the opposite direction (all P<0.05). In addition, TC, TG, LDL-c, non-HDL-c, TC/HDL-c, TG/HDL-c, LDL-c/HDL-c, and non-HDL-c/HDL-c had a positive relationship with the baPWV value, while HDL-c was on the contrary (all P <0.05). Finally, individuals with high non-HDL-c/HDL-c (OR 1.71, 95% CI 1.06-2.55, P = 0.013) and low HDL-c (OR 0.57, 95% CI 0.35-0.96, P = 0.024) were seem to be at high risk of arterial stiffness. CONCLUSIONS: As a risk indicator, non-HDL-c/HDL-c, which could be readily obtained from routine serum lipids, was significantly associated with baPWV. Non-HDL-c/HDL-c was superior to traditional lipid variables for estimating arterial stiffness in the middle-aged and elderly Chinese population.

Vulnerability for apoptosis in the hippocampal dentate gyrus of STZ-induced diabetic rats with cognitive impairment.

BACKGROUND: Hyperglycemia impaired hippocampal network via triggering suicide program of immanent neurons, this is regarded as an etiological factor for diabetic cognition deficits. AIM: To investigate the occurrence of apoptosis in the hippocampal dentate gyrus of streptozotocin (STZ)-induced diabetic rats with cognitive impairment and assess the gene and protein expression of the apoptotic proteins bax, bcl-2, and caspase-3. MATERIALS AND METHODS: Four weeks after the verification of STZ-induced diabetes, diabetic rats with and without cognitive decline subgroups were subsequently assigned according to Morris water maze test. The expression levels of apoptotic proteins were measured using real-time RT-PCR and western blotting, respectively. Neuronal apoptosis was detected by TUNEL staining and electron microscopy. RESULTS: In the dentate gyrus of the rats with cognitive decline, Bcl-2 exhibited lower gene and protein levels, whereas a higher expression of bax was detected contributing to a significant increase in their mean bax/bcl-2 ratio. However, caspase-3 was not activated. Statistically different numbers of TUNEL-staining cells and features of apoptosis were no found. CONCLUSIONS: The higher bax/bcl ratio probably represents neurons of dentate gyrus vulnerable to apoptosis in the diabetes with cognitive decline. However, the normal caspase-3 level suggests that apoptosis is not active in this illness phase.

PPARγ-mediated advanced glycation end products regulate neural stem cell proliferation but not neural differentiation through the BDNF-CREB pathway.

AIMS: To investigate the roles of PPARγ in advanced glycation end product (AGE)-mediated characteristics of neural stem cells (NSCs) and the molecular mechanisms of action. METHODS: We prepared pLentiLox3.7 lentiviral vectors expressing short hairpin RNA (shRNA) against PPARγ and transduced NSCs. MTT absorbance and cell counts were used to assay cell growth, and cell differentiation was analysed by confocal laser-scanning and western blots for the expression of MAP2/nestin. The protein and gene expression of the BDNF-CREB pathway components were examined by western blotting and real-time PCR. RESULTS: Immunoblot analysis indicated that shRNA delivered by lentiviral vectors silenced PPARγ expression in NSCs. The proliferation of NSCs and expression of BDNF pathway components dropped in AGE-BSA culture medium (400 mg/L and 200 mg/L) on Day 3 and Day 7, respectively (all P<0.001). PPARγ-silenced NSCs exhibited a significant increase in cell growth and expression of BDNF pathway components compared with NSCs incubated with AGE-BSA (all P<0.001). Immunocytochemistry and western blotting analysis showed that AGE-BSA (400 mg/L) induced a significant decrease in the expression of MAP2 both in NSCs and PPARγ-silenced NSCs, as standardised by nestin. There was no significant difference between NSCs and PPARγ-silenced NSCs in the presence of AGE-BSA. CONCLUSIONS: PPARγ plays roles in the AGE-mediated regulation of NSC proliferation but not neural differentiation through the BDNF-CREB pathway.

Meticillin-resistant Staphylococcus aureus isolated from foot ulcers in diabetic patients in a Chinese care hospital: risk factors for infection and prevalence.

A retrospective case-control study of 118 (male : female, 68 : 50) Chinese type 2 diabetic patients with foot ulcers (Wagner's grade 3-5) was conducted to determine the prevalence and risk factors for meticillin-resistant Staphylococcus aureus (MRSA) infection in relation to the original community or hospital parameters. Ulcer specimens were processed for Gram staining, aerobic culture and antimicrobial susceptibility testing. Staphylococcus species were tested for meticillin resistance using oxacillin. S. aureus was the most frequent pathogen (25.6 %) in diabetic patient specimens (160 isolates), and a high proportion of S. aureus isolates were MRSA (63.4 %). A high percentage of S. aureus isolates (65.4 %) satisfied the definition for hospital-associated MRSA (HA-MRSA) infection. The size of ulcers [adjusted odds ratio (OR) 1.61; 95 % confidence interval (CI) 1.22-2.12] and osteomyelitis (adjusted OR 18.51, 95 % CI 2.50-137.21) were independent predictors of MRSA infection. The HA-MRSA group had a significantly different distribution from the community-associated MRSA group with respect to age, history of diabetes and length of hospital stay (all P<0.001). Neuropathy, vascular disease (all P=0.049) and osteomyelitis (P=0.026) were the most common underlying conditions observed in the HA-MRSA group. This study contributes to the establishment of precautions against the emergence of MRSA including MRSA acquired from different sources among the Chinese population with diabetic foot ulcers based on their original or clinical parameters.