Yuexiu Wu

MicroRNA-200b Regulates Vascular Endothelial Growth Factor–Mediated Alterations in Diabetic Retinopathy

OBJECTIVE Diabetic retinopathy (DR) is a leading cause of blindness. Increased vascular endothelial growth factor (VEGF), promoting angiogenesis and increased permeability, is a key mechanistic abnormality in DR. We investigated microRNA (miRNA) alterations in DR with specific focus on miR-200b, and its downstream target, VEGF. RESEARCH DESIGN AND METHODS miRNA expression profiling microarray was used to examine the retinas of streptozotocin-induced diabetic rats. Expressions of specific miRNAs were verified with PCR in the rat retina and in glucose-exposed endothelial cells. A target search, based on sequence complementarities, identified specific targets. We analyzed mRNA levels and protein expression in endothelial cells from large vessels and retinal capillaries and in the rat retina, with or without injection of miR-200b mimic or antagomir. Localization of miR-200b and its functional analysis in the rat and human retinas were performed. RESULTS Alteration of several miRNAs, including downregulation of miR-200b, were observed in the retina in diabetes. Such downregulation was validated in the retina of diabetic rats and in endothelial cells incubated in glucose. In parallel, VEGF (target of miR-200b) mRNA and protein were elevated. In the retina, miR-200b was localized in neuronal, glial, and vascular elements. Transfection of endothelial cells and intravitreal injection of miR-200b mimic prevented diabetes-induced increased VEGF mRNA and protein. Also prevented were glucose-induced increased permeability and angiogenesis. Furthermore, transfection of miR-200b antagonists (antagomir) led to increased VEGF production. Similar alterations were seen in the human retina. CONCLUSIONS These studies show a novel mechanism involving miR-200b in DR. Identification of such mechanisms may lead to the development of novel miRNA-based therapy.

miR-146a–Mediated Extracellular Matrix Protein Production in Chronic Diabetes Complications

OBJECTIVE microRNAs (miRNAs), through transcriptional regulation, modulate several cellular processes. In diabetes, increased extracellular matrix protein fibronectin (FN) production is known to occur through histone acetylator p300. Here, we investigated the role of miR-146a, an FN-targeting miRNA, on FN production in diabetes and its relationship with p300. RESEARCH DESIGN AND METHODS miR-146a expressions were measured in endothelial cells from large vessels and retinal microvessels in various glucose levels. FN messenger RNA expression and protein levels with or without miR-146a mimic or antagomir transfection were examined. A luciferase assay was performed to detect miR-146a’s binding to FN 3′–untranslated region (UTR). Likewise, retinas from type 1 diabetic rats were studied with or without an intravitreal injection of miR-146a mimic. In situ hybridization was used to localize retinal miR-146a. Cardiac and renal tissues were analyzed from type 1 and type 2 diabetic animals. RESULTS A total of 25 mmol/L glucose decreased miR-146a expression and increased FN expression compared with 5 mmol/L glucose in both cell types. miR-146a mimic transfection prevented such change, whereas miR-146a antagomir transfection in the cells in 5 mmol/L glucose caused FN upregulation. A luciferase assay confirmed miR-146a’s binding to FN 3′-UTR. miR-146a was localized in the retinal endothelial cells and was decreased in diabetes. Intravitreal miR-146a mimic injection restored retinal miR-146a and decreased FN in diabetes. Additional experiments showed that p300 regulates miR-146a. Similar changes were seen in the retinas, kidneys, and hearts in type 1 and type 2 diabetic animals. CONCLUSIONS These studies showed a novel, glucose-induced molecular mechanism in which miR-146a participates in the transcriptional circuitry regulating extracellular matrix protein production in diabetes.

Preventive effects of North American ginseng (Panax quinquefolium) on diabetic nephropathy.

PURPOSE Ginseng has been used as an herbal medicine and nutritional supplement in East Asia for thousands of years and gained popularity in the west because of its various pharmacological properties. Panax ginseng (Asian ginseng) and Panax quinquefolium (North American ginseng) both are reported to possess antihyperglycemic properties. The aim of the present study is to evaluate the preventive effects of North American ginseng on diabetic nephropathy (DN) and the underlying mechanisms of such effects. METHODS Models of both type 1 (C57BL/6 mice with STZ-induced diabetes) and type 2 diabetes (db/db mice) and age- and sex-matched controls were examined. Alcoholic ginseng root (200mg/kgbodywt, daily oral gavage) extract was administered to the diabetic mice (type 1 and type 2) for two or four months in order to evaluate its effects on DN. RESULTS Dysmetabolic state in the diabetic mice was significantly improved by ginseng treatment. In the kidneys of diabetic animals, ginseng significantly prevented oxidative stress and reduced the NF-κB (p65) levels. Diabetes-induced up-regulations of ECM proteins and vasoactive factors in the kidneys were significantly diminished by ginseng administration. Furthermore, albuminuria and mesangial expansion in the diabetic mice were prevented by ginseng therapy. CONCLUSION North American ginseng has preventive effects on DN and it works through a combination of mechanisms such as antihyperglycemic and antioxidant activities.

Preventive effects of North American Ginseng (Panax quinquefolius) on Diabetic Retinopathy and Cardiomyopathy

Ginseng (Araliaceae) has multiple pharmacological actions because of its diverse phytochemical constituents. The aims of the present study are to evaluate the preventive effects of North American ginseng on diabetic retinopathy and cardiomyopathy and to delineate the underlying mechanisms of such effects. Models of both type 1 (C57BL/6 mice with streptozotocin‐induced diabetes) and type 2 diabetes (db/db mice) and age‐matched and sex‐matched controls were examined. Alcoholic ginseng root (200 mg/kg body weight, daily oral gavage) extract was administered to groups of both type 1 and type 2 diabetic mice for 2 or 4 months. Dysmetabolic state in the diabetic mice was significantly improved by ginseng treatment. In both the heart and retina of diabetic animals, ginseng treatment significantly prevented oxidative stress and diabetes‐induced upregulations of extracellular matrix proteins and vasoactive factors. Ginseng treatment in the diabetic animals resulted in enhancement of stroke volume, ejection fraction, cardiac output, and left ventricle pressure during systole and diastole and diminution of stroke work. In addition, mRNA expressions of atrial natriuretic factor and brain natriuretic factor (molecular markers for cardiac hypertrophy) were significantly diminished in ginseng‐treated diabetic mice. These data indicate that North American ginseng prevents the diabetes‐induced retinal and cardiac biochemical and functional changes probably through inhibition of oxidative stress. Copyright

Cdc42 negatively regulates intrinsic migration of highly aggressive breast cancer cells.

The small GTPase Cdc42 has been implicated as an important regulator of cell migration. However, whether Cdc42 plays similar role in all cancer cells irrespective of metastatic potential remains poorly defined. Here, we show by using three different breast cancer cell lines with different metastatic potential, the role of Cdc42 in cell migration/invasion and its relationship with a number of downstream signaling pathways controlling cell migration. Small interfering RNA (siRNA)‐mediated knockdown of Cdc42 in two highly metastatic breast cancer cell lines (MDA‐MB‐231 and C3L5) resulted in enhancement, whereas the same in moderately metastatic (Hs578T) cell line resulted in inhibition of intrinsic cellular migration/invasion. Furthermore, Cdc42 silencing in MDA‐MB‐231 and C3L5 but not Hs578T cells was shown to be accompanied by increased RhoA activity and phosphorylation of protein kinase C (PKC)‐δ, extracellular signal regulated kinase1/2 (Erk1/2), and protein kinase A (PKA). Pharmacological inhibition of PKCδ, MEK‐Erk1/2, or PKA was shown to inhibit migration of both control and Cdc42‐silenced MDA‐MB‐231 cells. Furthermore, introduction of constitutively active Cdc42 was shown to decrease migration/invasion of MDA‐MB‐231 and C3L5 but increase migration/invasion of Hs578T cells. This decreased migration/invasion of MDA‐MB‐231 and C3L5 cells was also shown to be accompanied by the decrease in the phosphorylations of PKCδ, Erk1/2, and PKA. These results suggested that endogenous Cdc42 could exert a negative regulatory influence on intrinsic migration/invasion and some potentially relevant changes in phosphorylation of PKCδ, Erk1/2, and PKA of some aggressive breast cancer cells. J. Cell. Physiol. 227: 1399–1407, 2012.

American ginseng (Panax quinquefolius) prevents glucose-induced oxidative stress and associated endothelial abnormalities.

PURPOSE Ginseng (Araliaceae), demonstrates widespread biological effects because of its purported antioxidant and other properties. The present study was undertaken to investigate the effects of American ginseng root extract on glucose-induced oxidative stress and associated oxidative damage to human umbilical vein endothelial cells (HUVECs). METHODS Following pretreatment with various concentrations of ginseng (alcoholic extract), HUVECs were incubated with various concentrations of d-glucose ranging from 5 to 25mmol/l for 24h. l-Glucose was used at a concentration of 25mmol/l as a control. RESULTS Glucose-induced oxidative stress detected by intracellular reactive oxygen species accumulation, superoxide anion generation and DNA damage in HUVECs were significantly prevented by ginseng. Treatment of HUVECs with ginseng further led to significant prevention of glucose-induced NF-κB activation. Glucose-induced increase in fibronectin (FN), EDB(+)FN (a splice variant of FN), endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF) mRNAs and protein levels were also prevented by ginseng treatment. CONCLUSION These data indicate that American ginseng prevented glucose-induced damage in the HUVECs through its antioxidant properties.

ERK5 Regulates Glucose-Induced Increased Fibronectin Production in the Endothelial Cells and in the Retina in Diabetes

PURPOSE Fibronectin (FN) production and deposition in the tissue is a characteristic feature of diabetic retinopathy. ERK5 is a recent member of the mitogen activated protein kinase (MAPK) family, which plays a critical role in cardiovascular development and maintaining endothelial cell integrity. The aim of this study was to investigate the role of ERK5 signaling in glucose-induced FN overproduction. METHODS Dermal-derived human microvascular endothelial cells (HMVECs) and human retinal microvascular endothelial cells (HRMECs) were used in this study. FN mRNA levels and secreted FN protein levels were measured using real-time PCR and ELISA, respectively. Constitutively active MAPK/ERK kinase 5 (MEK5 [CAMEK5]) adenovirus was used to upregulate ERK5. Dominant negative MEK5 (DNMEK5) and ERK5 siRNA (siERK5) were used to downregulate ERK5. Parallel retinal tissues of diabetic rats were examined. RESULTS A significant decrease of FN was observed at both protein and mRNA levels following CAMEK5 transduction in basal as well as in high glucose. DNMEK5 transduction led to further enhancement of glucose-induced increased FN expression. siERK5 treatment led to an increase of FN synthesis. Retinal tissues of diabetic rats showed FN upregulation and ERK5 downregulation. TGFβ1 mRNA and phosphorylated Smad2 were markedly suppressed by CAMEK5 transduction with and without glucose treatment. On the other hand, siERK5 transfection enhanced TGFβ1 mRNA expression. Exogenous nerve growth factor supplementation resulted in elevated phosphorylated and total ERK5 with and without glucose treatment. CONCLUSIONS Our experiments demonstrated a novel mechanism of glucose-induced increased FN production in diabetic retinopathy, which is mediated through decreased ERK5 signaling.

Modulation of ERK5 Is a Novel Mechanism by Which Cdc42 Regulates Migration of Breast Cancer Cells

Members of Rho family GTPases including Cdc42 are known to play pivotal roles in cell migration. Cell migration is also known to be regulated by many protein kinases. Kinetworks KPSS 11.0 phospho‐site screening of Cdc42‐silenced Hs578T breast cancer cells revealed most dramatic change in ERK5 MAP kinase. In the present study, we set out to determine the relationship between Cdc42 and ERK5 and its significance in breast cancer cell migration and invasion. Specific siRNAs were used for knocking down Cdc42 or ERK5 in breast cancer cells. Increased ERK5 phosphorylation in breast cancer cells was achieved by infection of constitutively active MEK5 adenovirus. The cells were then subjected to cell migration or invasion assay without the presence of serum or any growth factor. We found that Cdc42 negatively regulated phosphorylation of ERK5, which in turn exhibited an inverse relationship with migration and invasiveness of breast cancer cells. To find out some in vivo relevance of the results of our in vitro experiments we also examined the expression of ERK5 in the breast cancer tissues and their adjacent normal control tissues by real‐time RT‐PCR and immunocytochemistry. ERK5 expression was found to be reduced in breast cancer tissues as compared with their adjacent uninvolved mammary tissues. Therefore, Cdc42 may promote breast cancer cell migration and invasion by inhibiting ERK5 phosphorylation and ERK5 expression may be inversely correlated with the progression of some breast tumors. J. Cell. Biochem. 116: 124–132, 2015.

ERK5 Contributes to VEGF Alteration in Diabetic Retinopathy

Diabetic retinopathy is one of the most common causes of blindness in North America. Several signaling mechanisms are activated secondary to hyperglycemia in diabetes, leading to activation of vasoactive factors. We investigated a novel pathway, namely extracellular signal regulated kinase 5 (ERK5) mediated signaling, in modulating glucose-induced vascular endothelial growth factor (VEGF) expression. Human microvascular endothelial cells (HMVEC) were exposed to glucose. In parallel, retinal tissues from streptozotocin-induced diabetic rats were examined after 4 months of follow-up. In HMVECs, glucose caused initial activation followed by deactivation of ERK5 and its downstream mediators myocyte enhancing factor 2C (MEF2C) and Kruppel-like factor 2 (KLF2) mRNA expression. ERK5 inactivation further led to augmented VEGF mRNA expression. Furthermore, siRNA mediated ERK5 gene knockdown suppressed MEF2C and KLF2 expression and increased VEGF expression and angiogenesis. On the other hand, constitutively active MEK5, an activator of ERK5, increased ERK5 activation and ERK5 and KLF2 mRNA expression and attenuated basal- and glucose-induced VEGF mRNA expression. In the retina of diabetic rats, depletion of ERK5, KLF2 and upregulation of VEGF mRNA were demonstrated. These results indicated that ERK5 depletion contributes to glucose induced increased VEGF production and angiogenesis. Hence, ERK5 may be a putative therapeutic target to modulate VEGF expression in diabetic retinopathy.