Chi Fai Ng

Dipeptidyl Peptidase 4 Inhibitor Sitagliptin Protects Endothelial Function in Hypertension Through a Glucagon–Like Peptide 1–Dependent Mechanism

Sitagliptin, a selective dipeptidyl peptidase 4 inhibitor, inhibits the inactivation and degradation of glucagon like peptide 1 (GLP-1), which is used for the treatment of type 2 diabetes mellitus. However, little is known about the role of GLP-1 in hypertension. This study investigated whether the activation of GLP-1 signaling protects endothelial function in hypertension. Two-week sitagliptin treatment (10 mg/kg per day, oral gavage) improved endothelium-dependent relaxation in renal arteries, restored renal blood flow, and reduced systolic blood pressure in spontaneously hypertensive rats. In vivo sitagliptin treatment elevated GLP-1 and GLP-1 receptor expressions, increased cAMP level, and subsequently activated protein kinase A, liver kinase B1, AMP-activated protein kinase-&agr; and endothelial NO synthase in spontaneously hypertensive rat renal arteries. Inhibition of GLP-1 receptor, adenylyl cyclase, protein kinase A, AMP-activated protein kinase-&agr;, or NO synthase reversed the protective effects of sitagliptin. We also demonstrate that GLP-1 receptor agonist exendin 4 in vitro treatment had similar vasoprotective effects in spontaneously hypertensive rat renal arteries and increased NO production in spontaneously hypertensive rat aortic endothelial cells. Studies using transient expressions of wild-type and dominant-negative AMP-activated protein kinase-&agr;2 support the critical role of AMP-activated protein kinase-&agr; in mediating the effect of GLP-1 in endothelial cells. Ex vivo exendin 4 treatment also improved endothelial function of renal arteries from hypertensive patients. Our results elucidate that upregulation of GLP-1 and related agents improve endothelial function in hypertension by restoring NO bioavailability, suggesting that GLP-1 signaling could be a therapeutic target in hypertension-related vascular events.

Calcitriol protects renovascular function in hypertension by down-regulating angiotensin II type 1 receptors and reducing oxidative stress

AIMSnThe present study investigated whether or not calcitriol, an active form of vitamin D, protects against renovascular dysfunction in hypertension and, if so, whether or not such protection alters the expression of key proteins involved in that dysfunction.nnnMETHODS AND RESULTSnChanges in isometric tension showed that the impaired endothelium-dependent relaxations in renal arteries of hypertensive patients were enhanced by 12 h in vitro treatment with calcitriol. Dihydroethidium fluorescence revealed an elevated level of reactive oxygen species (ROS) in these arteries which was reduced by calcitriol. Immunofluorescence showed that calcitriol treatment reduced the expression of AT(1)R, NOX-2, NOX-4, and p67(phox) and increased that of superoxide dismutase (SOD)-1. Twelve-hour exposure to calcitriol prevented angiotensin (Ang) II-induced increases in ROS and the over-expression of NOX-2, NOX-4, and p67(phox) in renal arteries from normotensive patients. A specific antagonist of the human vitamin D receptor (VDR), TEI-9647, abolished these effects of calcitriol. Both in vitro exposure to and chronic in vivo administration of calcitriol enhanced relaxations to acetylcholine and abolished exaggerated endothelium-dependent contractions in renal arteries of normotensive rats pre-exposed to Ang II or harvested from spontaneously hypertensive rats (SHR). Reactive oxygen species levels and expressions of AT(1)R, NAD(P)H oxidase subunits, SOD-1, and SOD-2 in SHR arteries were normalized by the chronic treatment with calcitriol.nnnCONCLUSIONnIn vivo and in vitro activation of VDR with calcitriol improves endothelial function by normalizing the expressions of AT(1)R and radical generating and scavenging enzymes and thus preventing ROS over-production. The present findings suggest that calcitriol is effective in preserving endothelial function in hypertension.

ERRα augments HIF‐1 signalling by directly interacting with HIF‐1α in normoxic and hypoxic prostate cancer cells

Adaptation of cancer cells to a hypoxic microenvironment is important for their facilitated malignant growth and advanced development. One major mechanism mediating the hypoxic response involves up‐regulation of hypoxia‐inducible factor 1 (HIF‐1) expression, which controls reprogramming of energy metabolism and angiogenesis. Oestrogen‐related receptor‐α (ERRα) is a pivotal regulator of cellular energy metabolism and many biosynthetic pathways, and has also been proposed to be an important factor promoting the Warburg effect in advanced cancer. We and others have previously shown that ERRα expression is increased in prostate cancer and is also a prognostic marker. Here we show that ERRα is oncogenic in prostate cancer and also a key hypoxic growth regulator. ERRα‐over‐expressing prostate cancer cells were more resistant to hypoxia and showed enhanced HIF‐1α protein expression and HIF‐1 signalling. These effects could also be observed in ERRα‐over‐expressing cells grown under normoxia, suggesting that ERRα could function to pre‐adapt cancer cells to meet hypoxia stress. Immunoprecipitation and FRET assays indicated that ERRα could physically interact with HIF‐1α via its AF‐2 domain. A ubiquitination assay showed that this ERRα–HIF‐1α interaction could inhibit ubiquitination of HIF‐1α and thus reduce its degradation. Such ERRα–HIF‐1α interaction could be attenuated by XCT790, an ERRα‐specific inverse agonist, resulting in reduced HIF‐1α levels. In summary, we show that ERRα can promote the hypoxic growth adaptation of prostate cancer cells via a protective interaction with HIF‐1α, suggesting ERRα as a potential therapeutic target for cancer treatment. Copyright

Unconjugated bilirubin mediates heme oxygenase-1-induced vascular benefits in diabetic mice

Heme oxygenase-1 (HO-1) exerts vasoprotective effects. Such benefit in diabetic vasculopathy, however, remains unclear. We hypothesize that bilirubin mediates HO-1–induced vascular benefits in diabetes. Diabetic db/db mice were treated with hemin (HO-1 inducer) for 2 weeks, and aortas were isolated for functional and molecular assays. Nitric oxide (NO) production was measured in cultured endothelial cells. Hemin treatment augmented endothelium-dependent relaxations (EDRs) and elevated Akt and endothelial NO synthase (eNOS) phosphorylation in db/db mouse aortas, which were reversed by the HO-1 inhibitor SnMP or HO-1 silencing virus. Hemin treatment increased serum bilirubin, and ex vivo bilirubin treatment improved relaxations in diabetic mouse aortas, which was reversed by the Akt inhibitor. Biliverdin reductase silencing virus attenuated the effect of hemin. Chronic bilirubin treatment improved EDRs in db/db mouse aortas. Hemin and bilirubin reversed high glucose–induced reductions in Akt and eNOS phosphorylation and NO production. The effect of hemin but not bilirubin was inhibited by biliverdin reductase silencing virus. Furthermore, bilirubin augmented EDRs in renal arteries from diabetic patients. In summary, HO-1–induced restoration of endothelial function in diabetic mice is most likely mediated by bilirubin, which preserves NO bioavailability through the Akt/eNOS/NO cascade, suggesting bilirubin as a potential therapeutic target for clinical intervention of diabetic vasculopathy.

Inhibition of miR-200c restores endothelial function in diabetic mice through suppression of COX-2

Endothelial dysfunction plays a crucial role in the development of diabetic vasculopathy. Our initial quantitative PCR results showed an increased miR-200c expression in arteries from diabetic mice and patients with diabetes. However, whether miR-200c is involved in diabetic endothelial dysfunction is unknown. Overexpression of miR-200c impaired endothelium-dependent relaxations (EDRs) in nondiabetic mouse aortas, whereas suppression of miR-200c by anti–miR-200c enhanced EDRs in diabetic db/db mice. miR-200c suppressed ZEB1 expression, and ZEB1 overexpression ameliorated endothelial dysfunction induced by miR-200c or associated with diabetes. More importantly, overexpression of anti–miR-200c or ZEB1 in vivo attenuated miR-200c expression and improved EDRs in db/db mice. Mechanistic study with the use of COX-2−/− mice revealed that COX-2 mediated miR-200c–induced endothelial dysfunction and that miR-200c upregulated COX-2 expression in endothelial cells through suppression of ZEB1 and increased production of prostaglandin E2, which also reduced EDR. This study demonstrates for the first time to our knowledge that miR-200c is a new mediator of diabetic endothelial dysfunction and inhibition of miR-200c rescues EDRs in diabetic mice. These new findings suggest the potential usefulness of miR-200c as the target for drug intervention against diabetic vascular complications.

Orphan nuclear receptor TLX functions as a potent suppressor of oncogene-induced senescence in prostate cancer via its transcriptional co-regulation of the CDKN1A (p21WAF1/CIP1) and SIRT1 genes

Oncogene‐induced senescence is an important tumour‐suppressing mechanism to prevent both premalignant transformation and cancer progression. Overcoming this process is a critical step in early cancer development. The druggable orphan nuclear receptor TLX (NR2E1) is characterized as an important regulator of neural stem cells and is also implicated in the development of some brain tumours. However, its exact functional roles in cancer growth regulation still remain unclear. Here we report that TLX can act as a promoter of tumourigenesis in prostate cancer by suppressing oncogene‐induced senescence. We determined that TLX exhibited an increased expression in high‐grade prostate cancer tissues and many prostate cancer cell lines. Functional studies revealed that TLX could perform an oncogenic function in prostate cancer cells, as its knockdown triggered cellular senescence and cell growth arrest in vitro and in vivo, whereas its over‐expression promoted the malignant growth of prostate cancer cells. Furthermore, enhancement of TLX activity, by either ectopic expression or ligand stimulation, could potently prevent doxorubicin‐induced senescence in prostate cancer cells and also allow prostatic epithelial cells to escape oncogene‐induced senescence induced either by activated oncogene H‐RasG12V or knockdown of tumour suppressor PTEN, via a mechanism of direct but differential transcriptional regulation of two senescence‐associated genes, repression of CDKN1A and transactivation of SIRT1. Together, our present study shows, for the first time, that TLX may play an important role in prostate carcinogenesis through its suppression of oncogene‐induced senescence, and also suggests that targeting the senescence‐regulatory TLX is of potential therapeutic significance in prostate cancer. Copyright

Calcitriol restores renovascular function in estrogen-deficient rats through downregulation of cyclooxygenase-2 and the thromboxane-prostanoid receptor

Cardiovascular risks increase in postmenopausal women. While vitamin D is supplemented for osteoporosis, it is not known whether it protects renal arterial function during estrogen deficiency. Here we measured changes in renovascular reactivity induced by ovariectomy in rats and examined whether calcitriol, the most active form of vitamin D, was able to correct such changes. The impairment of endothelium-dependent relaxation in renal arteries from ovariectomized rats was effectively reversed by long-term calcitriol treatment. It was also corrected by acute exposure to cyclooxygenase-2 (COX-2) inhibitors and a thromboxane-prostanoid receptor antagonist, respectively. Calcitriol normalized the overexpression of COX-2 and thromboxane-prostanoid receptors in intralobal renal artery segments and aortic endothelial cells isolated from ovariectomized rats. In vitro exposure of the arterial segments to calcitriol for 12u2009h improved relaxation and downregulated thromboxane-prostanoid receptors. The attenuated nitric oxide production in ovariectomized rat aortic endothelial cells was restored following a 12-h treatment with calcitriol, COX-2 inhibition, or thromboxane-prostanoid receptor antagonism. Thus, impaired endothelium-dependent renal artery relaxation in ovariectomized rats is mediated largely through increased activity and expression of COX-2 and the thromboxane-prostanoid receptor. Calcitriol restores endothelial function through downregulating both signaling proteins during estrogen deficiency.

Ion channel TRPM8 promotes hypoxic growth of prostate cancer cells via an O2‐independent and RACK1‐mediated mechanism of HIF‐1α stabilization

The growth adaptation of cancer cells to a hypoxic tumour microenvironment is mostly regulated by hypoxia‐induced transcription factor HIF‐1. HIF‐1 transcriptional activity is strictly controlled by protein levels of the HIF‐1α subunit, which is tightly regulated by a well‐characterized O2‐dependent ubiquitin ligase–proteasomal degradation pathway. The cold‐sensitive Ca2+ channel protein TRPM8 exhibits increased expression in advanced prostate cancer. However, its exact functional roles in prostate cancer growth regulation are unclear and controversial. In this work, we show that TRPM8 promotes in vitro hypoxic growth capacities, drug resistance, and in vivo tumourigenicity, accompanied with enhanced HIF‐1α protein levels. These effects are further potentiated by TRPM8 agonists but suppressed by TRPM8 gene knockdown and blocking with antagonists or TRPM8 antibody. TRPM8‐induced suppression of HIF‐1α ubiquitination and enhanced HIF‐1 transactivation were attenuated by forced RACK1 expression and TRPM8 overexpression reduced phospho‐RACK1 levels, thus affecting its dimerization status, and promoted RACK1 binding to HIF‐1α and calcineurin. These data indicate that TRPM8‐induced increase of HIF‐1α protein in hypoxia‐ or normoxia‐exposed prostate cancer cells was mediated through a newly characterized Ca2+‐dependent but O2‐independent mechanism involving binding of RACK1 to HIF‐1α and RACK1‐mediated ubiquitination of HIF‐1α. Collectively, our study not only provides a mechanistic insight into how TRPM8 promotes the hypoxic growth adaptation of cancer cells via its promotion of RACK1‐mediated stabilization of HIF‐1α but also suggests a potential therapeutic strategy for prostate cancer by targeting TRPM8.Copyright

Ginsenoside Rb3 attenuates oxidative stress and preserves endothelial function in renal arteries from hypertensive rats

Panax ginseng is commonly used to treat cardiovascular conditions in Oriental countries. This study investigated the mechanisms underlying the vascular benefits of ginsenoside Rb3 (Rb3) in hypertension.

Increased expression of activated endothelial nitric oxide synthase contributes to antiandrogen resistance in prostate cancer cells by suppressing androgen receptor transactivation

Development of antiandrogen-resistance in advanced prostate cancer involves multiple androgen receptor (AR)-dependent and -independent pathways. Here, we demonstrated that endothelial nitric oxide synthase (eNOS) exhibited an overexpression pattern in hormone-refractory prostate cancer and several models of advanced hormone-resistant prostate cancer. We further established a novel in vitro model of antiandrogen-resistant prostate cancer (LNCaP-BC) by long-term bicalutamide treatment. Besides antiandrogen-resistant and other enhanced malignant growth phenotypes, LNCaP-BC cells exhibited an increased activated eNOS expression and NO production, and suppressed AR transactivation status. Treatment with a NOS inhibitor L-NAME could re-sensitize the growth response to bicalutamide and enhance the AR transactivation in LNCaP-BC cells. Together, our present findings indicate that increased NO production by acquired increased expression of activated eNOS could contribute to the antiandrogen-resistant growth of prostate cancer cells, via a mechanism of NO-mediated suppression of AR activity, and also targeting eNOS could be a potential therapeutic strategy for antiandrogen-resistant prostate cancer.