Effect of long-term glycemic variability on estimated glomerular filtration rate decline among patients with type 2 diabetes mellitus: Insights from the Diabetic Nephropathy Cohort in Singapore.

BACKGROUND: In the present study, we examined the association between HbA1c variability and renal disease progression based on estimated glomerular filtration rate (eGFR) decline in patients with type 2 diabetes mellitus (T2DM) in Singapore. METHODS: Glycemic burden and renal function were retrospectively assessed in 1628 patients in 2002-2014. Multivariable logistic regression was used to assess the relationships between HbA1c variability (expressed as HbA1c coefficient of variation [HbA1c-CV] in quartiles), HbA1c intrapersonal mean (HbA1c-IM), and eGFR decline, adjusted for baseline covariates. RESULTS: Among patients with relatively good glycemic control (i.e. HbA1c-IM below the median cohort value [8.0%]), HbA1c-CV Quartile 4 was associated with eGFR decline (odds ratio [OR] 1.88; 95% confidence interval [CI] 1.10-3.25). The OR for HbA1c-CV Quartile 4 was 2.20 (95% CI 1.24-3.89) after additional adjustment for HbA1c-IM. Where HbA1c-IM was above the median cohort value, HbA1c-CV Quartiles 3 and 4 were associated with eGFR decline, with ORs of 2.60 (95% CI 1.48-4.55) and 3.29 (95% CI 1.89-5.76) respectively. After further adjusting for HbA1c-IM, the ORs for Quartiles 3 and 4 were 2.69 (95% CI 1.53-4.74) and 3.51 (95% CI 1.98-6.21), respectively. CONCLUSIONS: Variability in HbA1c is strongly and independently associated with eGFR decline in patients with T2DM independent of mean HbA1c. The findings may highlight the importance of sustained stable glycemic control in management of diabetes mellitus.

Heart of glass anchors Rasip1 at endothelial cell-cell junctions to support vascular integrity.

Heart of Glass (HEG1), a transmembrane receptor, and Rasip1, an endothelial-specific Rap1-binding protein, are both essential for cardiovascular development. Here we performed a proteomic screen for novel HEG1 interactors and report that HEG1 binds directly to Rasip1. Rasip1 localizes to forming endothelial cell (EC) cell-cell junctions and silencing HEG1 prevents this localization. Conversely, mitochondria-targeted HEG1 relocalizes Rasip1 to mitochondria in cells. The Rasip1-binding site in HEG1 contains a 9 residue sequence, deletion of which abrogates HEG1's ability to recruit Rasip1. HEG1 binds to a central region of Rasip1 and deletion of this domain eliminates Rasip1's ability to bind HEG1, to translocate to EC junctions, to inhibit ROCK activity, and to maintain EC junctional integrity. These studies establish that the binding of HEG1 to Rasip1 mediates Rap1-dependent recruitment of Rasip1 to and stabilization of EC cell-cell junctions.

Structural basis of the junctional anchorage of the cerebral cavernous malformations complex.

The products of genes that cause cerebral cavernous malformations (CCM1/KRIT1, CCM2, and CCM3) physically interact. CCM1/KRIT1 links this complex to endothelial cell (EC) junctions and maintains junctional integrity in part by inhibiting RhoA. Heart of glass (HEG1), a transmembrane protein, associates with KRIT1. In this paper, we show that the KRIT1 band 4.1, ezrin, radixin, and moesin (FERM) domain bound the HEG1 C terminus (K(d) = 1.2 µM) and solved the structure of this assembly. The KRIT1 F1 and F3 subdomain interface formed a hydrophobic groove that binds HEG1(Tyr(1,380)-Phe(1,381)), thus defining a new mode of FERM domain-membrane protein interaction. This structure enabled design of KRIT1(L717,721A), which exhibited a >100-fold reduction in HEG1 affinity. Although well folded and expressed, KRIT1(L717,721A) failed to target to EC junctions or complement the effects of KRIT1 depletion on zebrafish cardiovascular development or Rho kinase activation in EC. These data establish that this novel FERM-membrane protein interaction anchors CCM1/KRIT1 at EC junctions to support cardiovascular development.

Knockdown of lactate dehydrogenase A suppresses tumor growth and metastasis of human hepatocellular carcinoma.

In previous studies, lactate dehydrogenase A (LDHA) was identified as one of the leading genes that promote the proliferative and tumorigenic potential of malignancies. However, less definitive evidence was reported in hepatocellular carcinoma (HCC) cells. Furthermore, the role of LDHA in promoting metastasis of HCC, and its possible mechanism, is not clear. In this study, RNA interference (RNAi) mediated by lentiviral vectors (which induce strong down-regulation of gene expression) was used to analyze the role of LDHA in tumor growth and metastasis in HCC. We performed transient and stable RNAi knockdowns of LDHA in HCCLM3 cells, a line that over-expresses LDHA and has a high metastatic potential. Our studies reveal that previously unidentified effects of LHDA may mediate tumor growth and metastasic effects in HCC. First, HCC cell lines over-express LDHA. Second, LDHA inhibition results in increased apoptosis via production of reactive oxygen species in HCCLM3 cells. Thus, LDHA knockdown resulted in significant reduction in metastatic potential in a xenograft mouse model. Furthermore, we found that FAK, MMP-2, VEGF and E-cadherin proteins contribute to inhibitory effects on metastasis in HCC cells. These studies have important implications for understanding the mechanisms by which LDHA promotes tumor growth and metastasis.

A mechanism of Rap1-induced stabilization of endothelial cell--cell junctions.

Activation of Rap1 small GTPases stabilizes cell--cell junctions, and this activity requires Krev Interaction Trapped gene 1 (KRIT1). Loss of KRIT1 disrupts cardiovascular development and causes autosomal dominant familial cerebral cavernous malformations. Here we report that native KRIT1 protein binds the effector loop of Rap1A but not H-Ras in a GTP-dependent manner, establishing that it is an authentic Rap1-specific effector. By modeling the KRIT1-Rap1 interface we designed a well-folded KRIT1 mutant that exhibited a ~40-fold-reduced affinity for Rap1A and maintained other KRIT1-binding functions. Direct binding of KRIT1 to Rap1 stabilized endothelial cell-cell junctions in vitro and was required for cardiovascular development in vivo. Mechanistically, Rap1 binding released KRIT1 from microtubules, enabling it to locate to cell--cell junctions, where it suppressed Rho kinase signaling and stabilized the junctions. These studies establish that the direct physical interaction of Rap1 with KRIT1 enables the translocation of microtubule-sequestered KRIT1 to junctions, thereby supporting junctional integrity and cardiovascular development.

Regulation of cardiovascular development and integrity by the heart of glass-cerebral cavernous malformation protein pathway.

Cerebral cavernous malformations (CCMs) are human vascular malformations caused by mutations in three genes of unknown function: KRIT1, CCM2 and PDCD10. Here we show that the heart of glass (HEG1) receptor, which in zebrafish has been linked to ccm gene function, is selectively expressed in endothelial cells. Heg1(-/-) mice showed defective integrity of the heart, blood vessels and lymphatic vessels. Heg1(-/-); Ccm2(lacZ/+) and Ccm2(lacZ/lacZ) mice had more severe cardiovascular defects and died early in development owing to a failure of nascent endothelial cells to associate into patent vessels. This endothelial cell phenotype was shared by zebrafish embryos deficient in heg, krit1 or ccm2 and reproduced in CCM2-deficient human endothelial cells in vitro. Defects in the hearts of zebrafish lacking heg or ccm2, in the aortas of early mouse embryos lacking CCM2 and in the lymphatic vessels of neonatal mice lacking HEG1 were associated with abnormal endothelial cell junctions like those observed in human CCMs. Biochemical and cellular imaging analyses identified a cell-autonomous pathway in which the HEG1 receptor couples to KRIT1 at these cell junctions. This study identifies HEG1-CCM protein signaling as a crucial regulator of heart and vessel formation and integrity.

Differentiation of vascular myofibroblasts induced by transforming growth factor-beta1 requires the involvement of protein kinase Calpha.

In response to vascular injury, adventitial fibroblasts can modulate their phenotype to myofibroblasts, cells that participate in arterial remodeling. However, the signaling mechanisms underlying the vascular myofibroblast differentiation remain unknown. Since protein kinase C (PKC) is a key enzyme for cell differentiation, we examined whether PKC isoforms were involved in the vascular myofibroblast differentiation. The association between PKCalpha and myofibroblast differentiation was investigated in cultured rat aortic fibroblasts treated with transforming growth factor-beta1 (TGFbeta1). Confocal immunofluorescence microscopy indicated that fibroblasts expressed alpha-smooth muscle actin (alpha-SM actin) after TGFbeta1 treatment. Moreover, TGFbeta1 stimulation increased both PKCalpha mRNA expression (measured by real-time quantitative RT-PCR) and PKC activity (determined by histone-like pseudosubstrate phosphorylation) in adventitial fibroblasts. Western blot analysis indicated that PKCalpha protein expression was higher in TGFbeta1-treated fibroblasts than in untreated cells. TGFbeta1-induced expression of alpha-SM actin was inhibited in a dose-dependent manner by treating cells with a PKC inhibitor, calphostin C, and was abolished by depleting PKCalpha with antisense PKCalpha oligodeoxynucleotides. Our results demonstrate that TGFbeta1 induces adventitial myofibroblast differentiation via a PKCalpha-dependent process.