Empagliflozin lessened cardiac injury and reduced visceral adipocyte hypertrophy in prediabetic rats with metabolic syndrome.

BACKGROUND: The potential benefit of SGLT2 inhibitors in metabolic syndrome is with prediabetic stage unclear. This work was undertaken to investigate the non-glycemic effect of empagliflozin on metabolic syndrome rats with prediabetes. METHODS: SHR/NDmcr-cp(+/+) rats (SHRcp), a model of metabolic syndrome with prediabetes, were given empagliflozin for 10 weeks to examine the effects on urinary sodium and water balance, visceral and subcutaneous adipocyte, and cardiac injury. Further, the effect of empagliflozin on blood pressure and autonomic nervous system was continuously investigated by using radiotelemetry system. RESULTS: Empagliflozin significantly reduced urinary sodium and water balance of SHRcp only within 1 week of the treatment, but later than 1 week did not alter them throughout the treatment. Empagliflozin significantly reduced body weight of SHRcp, which was mainly attributed to the significant reduction of subcutaneous fat mass. Empagliflozin significantly reduced the size of visceral adipocytes and increased the number of smaller size of adipocytes, which was associated with the attenuation of oxidative stress. Empagliflozin ameliorated cardiac hypertrophy and fibrosis of SHRcp, in association with the attenuation of cardiac oxidative stress and inflammation. However, empagliflozin did not significantly change blood pressure, heart rate, sympathetic activity, or baroreceptor function, as evidenced by radiotelemetry analysis. CONCLUSIONS: Our present work provided the evidence that SGLT2 inhibition reduced visceral adipocytes hypertrophy and ameliorated cardiac injury in prediabetic metabolic syndrome rat, independently of diuretic effect or blood pressure lowering effect. Thus, SGLT2 inhibition seems to be a promising therapeutic strategy for prediabetic metabolic syndrome.

Assessment of the Correlations Between Brain Weight and Brain Edema in Experimental Subarachnoid Hemorrhage.

Because brain edema is correlated with poor outcome in clinical subarachnoid hemorrhage (SAH), appropriate evaluation methods for brain edema are important in experimental SAH studies. Although brain water content (BWC) is widely used to evaluate brain edema in stroke research, the usefulness of brain weight is undetermined. In this study, we examined the role of brain weight in the evaluation of brain edema in experimental SAH. The endovascular perforation model of SAH was used, and rats were assessed by neurological scoring (NS). The brains were quickly removed at 24 h after the operation, and the weights of wet cerebrum (WWC) and dry cerebrum (WDC) were measured to determine the brain water content (BWC). The correlations of those values with each other and to body weight (BW) were then examined to reveal the significance of brain weight. The rats were assigned to sham-operated (n = 8) and SAH (n = 16) groups. There were no significant differences in WWC between the groups (p = 0.61). WWC was correlated with BWC but not with NS in all rats. In addition, WWC was clearly correlated with BW and WDC, which is thought to substitute for the original brain weight. From these results, we suggest that the measurement of brain weight as an evaluation of brain edema is limited and that BW and original brain volume can be confounding factors in evaluation.

DPP-4 inhibition with linagliptin ameliorates cognitive impairment and brain atrophy induced by transient cerebral ischemia in type 2 diabetic mice.

BACKGROUND: It is unclear whether dipeptidylpeptidase-4 (DPP-4) inhibition can counteract the impairment of cognitive function and brain injury caused by transient cerebral ischemia in type 2 diabetes. The present study was undertaken to test our hypothesis that linagliptin, a DPP-4 inhibitor, administration following transient cerebral ischemia can ameliorate cognitive impairment and brain injury in diabetic mice. METHODS: db/db mice, a model of obese type 2 diabetes, were subjected to transient cerebral ischemia by 17 min of bilateral common carotid artery occlusion (BCCAO), and were administered (1) vehicle or (2) linagliptin for 8 weeks or 1 week. For the long-term experiment on 8 weeks of linagliptin treatment, cognitive function, and volume and neuronal cell number of hippocampus and cortex were estimated in each group of mice. For the short-term experiment on 1 week of linagliptin treatment, cerebral IgG extravasation, Iba-1 positive cell number (reactive microglia), oxidative stress, and claudin-5 and gp91phox protein levels were measured in each group of mice. RESULTS: Linagliptin administration almost completely suppressed the circulating DPP-4 activity in db/db mice, but did not significantly reduce blood glucose or ameliorate glucose intolerance in db/db mice. Linagliptin administration following transient cerebral ischemia significantly counteracted cognitive impairment in diabetic mice, as estimated by water maze test and passive avoidance test. Linagliptin administration ameliorated the decrease in cerebral volume and neuronal cell number in hippocampus and cortex of diabetic mice. Linagliptin administration significantly reduced the increase in cerebral IgG extravasation and the increase in reactive microglia caused by transient cerebral ischemia in diabetic mice. Furthermore, linagliptin significantly suppressed the increase in cerebral oxidative stress in transient cerebral ischemia-subjected diabetic mice. Furthermore, linagliptin significantly increased cerebral claudin-5 and significantly decreased gp91phox in diabetic mice subjected to transient cerebral ischemia. CONCLUSIONS: DPP-4 inhibition with linagliptin counteracted cognitive impairment and brain atrophy induced by transient cerebral ischemia in diabetic mice, independently of blood glucose lowering effect. This cerebroprotective effect of linagliptin was associated with the suppression of blood-brain barrier disruption and the attenuation of cerebral oxidative stress. Thus, our present work highlights DPP-4 inhibition as a promising therapeutic strategy for cognitive impairment and cerebral vascular complications in type 2 diabetes.

Apoptosis signal-regulating kinase 1 is a novel target molecule for cognitive impairment induced by chronic cerebral hypoperfusion.

OBJECTIVE: There are currently no specific strategies for the treatment or prevention of vascular dementia. White matter lesions, a common pathology in cerebral small vessel disease, are a major cause of vascular dementia. We investigated whether apoptosis signal-regulating kinase 1 (ASK1) might be a key molecule in cerebral hypoperfusion, associated with blood-brain barrier breakdown and white matter lesions. APPROACH AND RESULTS: A mouse model of cognitive impairment was developed by inducing chronic cerebral hypoperfusion in white matter including the corpus callosum via bilateral common carotid artery stenosis (BCAS) surgery. BCAS-induced white matter lesions caused cognitive decline in C57BL/6J (wild-type) mice but not in ASK1-deficient (ASK1(-/-)) mice. Phosphorylated ASK1 increased in wild-type mouse brains, and phosphorylated p38 and tumor necrosis factor-α expression increased in corpus callosum cerebral endothelial cells after BCAS in wild-type mice but not in ASK1(-/-) mice. BCAS decreased claudin-5 expression and disrupted blood-brain barrier in the corpus callosum of wild-type but not ASK1(-/-) mice. Cerebral nitrotyrosine was increased in wild-type and ASK1(-/-) BCAS mice. Cerebral phosphorylated ASK1 did not increase in wild-type mice treated with NADPH-oxidase inhibitor. A p38 inhibitor and NADPH-oxidase inhibitor mimicked the protective effect of ASK1 deficiency against cognitive impairment. Specific ASK1 inhibitor prevented cognitive decline in BCAS mice. In vitro oxygen-glucose deprivation and tumor necrosis factor-α stimulation caused the disruption of endothelial tight junctions from wild-type mice but not ASK1(-/-) mice. CONCLUSIONS: Oxidative stress-ASK1-p38 cascade plays a role in the pathogenesis of cognitive impairment, through blood-brain barrier breakdown via the disruption of endothelial tight junctions. ASK1 might be a promising therapeutic target for chronic cerebral hypoperfusion-induced cognitive impairment.

Glycemic control with empagliflozin, a novel selective SGLT2 inhibitor, ameliorates cardiovascular injury and cognitive dysfunction in obese and type 2 diabetic mice.

BACKGROUND: There has been uncertainty regarding the benefit of glycemic control with antidiabetic agents in prevention of diabetic macrovascular disease. Further development of novel antidiabetic agents is essential for overcoming the burden of diabetic macrovascular disease. The renal sodium glucose co-transporter 2 (SGLT2) inhibitor is a novel antihyperglycemic agent for treatment of type 2 diabetes. This work was performed to determine whether empagliflozin, a novel SGLT2 inhibitor, can ameliorate cardiovascular injury and cognitive decline in db/db mouse, a model of obesity and type 2 diabetes. METHODS: (1) Short-term experiment: The first experiment was performed to examine the effect of 7 days of empagliflozin treatment on urinary glucose excretion and urinary electrolyte excretion in db/db mice. (2) Long-term experiment: The second experiment was undertaken to examine the effect of 10 weeks of empagliflozin treatment on cardiovascular injury, vascular dysfunction, cognitive decline, and renal injury in db/db mice. RESULTS: (1) Short-term experiment: Empagliflozin administration significantly increased urinary glucose excretion, urine volume, and urinary sodium excretion in db/db mice on day 1, but did not increase these parameters from day 2. However, blood glucose levels in db/db mice were continuously decreased by empagliflozin throughout 7 days of the treatment. (2) Long-term experiment: Empagliflozin treatment caused sustained decrease in blood glucose in db/db mice throughout 10 weeks of the treatment and significantly slowed the progression of type 2 diabetes. Empagliflozin significantly ameliorated cardiac interstitial fibrosis, pericoronary arterial fibrosis, coronary arterial thickening, cardiac macrophage infiltration, and the impairment of vascular dilating function in db/db mice, and these beneficial effects of empagliflozin were associated with attenuation of oxidative stress in cardiovascular tissue of db/db mice. Furthermore, empagliflozin significantly prevented the impairment of cognitive function in db/db mice, which was associated with the attenuation of cerebral oxidative stress and the increase in cerebral brain-derived neurotrophic factor. Empagliflozin ameliorated albuminuria, and glomerular injury in db/db mice. CONCLUSIONS: Glycemic control with empagliflozin significantly ameliorated cardiovascular injury and remodeling, vascular dysfunction, and cognitive decline in obese and type 2 diabetic mice. Thus, empagliflozin seems to be potentially a promising therapeutic agent for diabetic macrovascular disease and cognitive decline.

DPP-4 inhibitor linagliptin ameliorates cardiovascular injury in salt-sensitive hypertensive rats independently of blood glucose and blood pressure.

BACKGROUND: It remains to be elucidated whether dipeptidylpeptidase-4 (DPP-4) inhibitor can ameliorate cardiovascular injury in salt-sensitive hypertension. The present study was undertaken to test our hypothesis that linagliptin, a DPP-4 inhibitor, administration initiated after onset of hypertension and cardiac hypertrophy can ameliorate cardiovascular injury in Dahl salt-sensitive hypertensive rats (DS rats). METHODS: High-salt loaded DS rats with established hypertension and cardiac hypertrophy were divided into two groups, and were orally given (1) vehicle or (2) linagliptin (3 mg/kg/day) once a day for 4 weeks, and cardiovascular protective effects of linagliptin in DS rats were evaluated. RESULTS: Linagliptin did not significantly affect blood pressure and blood glucose levels in DS rats. Linagliptin significantly lessened cardiac hypertrophy in DS rats, as estimated by cardiac weight and echocardiographic parameters. Linagliptin significantly ameliorated cardiac fibrosis, cardiac macrophage infiltration, and coronary arterial remodeling in DS rats. Furthermore, linagliptin significantly mitigated the impairment of vascular function in DS rats, as shown by the improvement of acetylcholine-induced or sodium nitroprusside-induced vascular relaxation by linagliptin. These cardiovascular protective effects of linagliptin were associated with the attenuation of oxidative stress, NADPH oxidase subunits, p67phox and p22 phox, and angiotensin-converting enzyme (ACE). CONCLUSIONS: Our results provided the experimental evidence that linagliptin treatment initiated after the appearance of hypertension and cardiac hypertrophy protected against cardiovascular injury induced by salt-sensitive hypertension, independently of blood pressure and blood glucose. These beneficial effects of linagliptin seem to be attributed to the reduction of oxidative stress and ACE.

Rosuvastatin ameliorates early brain injury after subarachnoid hemorrhage via suppression of superoxide formation and nuclear factor-kappa B activation in rats.

BACKGROUND: Statins, or 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, have been suggested to possess pleiotropic effects, including antioxidant and anti-inflammatory properties. We investigated the protective effects of pretreatment with rosuvastatin, a relatively hydrophilic statin, on early brain injury (EBI) after a subarachnoid hemorrhage (SAH), using the endovascular perforation SAH model. METHODS: Eighty-six male Sprague-Dawley rats were randomly divided into 3 groups: (1) sham operation, (2) SAH+vehicle, and (3) SAH+10 mg/kg rosuvastatin. Rosuvastatin or vehicle was orally administered to rats once daily from 7 days before to 1 day after the SAH operation. After SAH, we examined the effects of rosuvastatin on the neurologic score, brain water content, neuronal cell death estimated by terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate nick end labeling staining, blood-brain barrier disruption by immunoglobulin G (IgG) extravasation, oxidative stress, and proinflammatory molecules. RESULTS: Compared with the vehicle group, rosuvastatin significantly improved the neurologic score and reduced the brain water content, neuronal cell death, and IgG extravasation. Rosuvastatin inhibited brain superoxide production, nuclear factor-kappa B (NF-κB) activation, and the increase in activated microglial cells after SAH. The increased expressions of tumor necrosis factor-alpha, endothelial matrix metalloproteinase-9, and neuronal cyclooxygenase-2 induced by SAH were prevented by rosuvastatin pretreatment. CONCLUSIONS: The present study demonstrates that rosuvastatin pretreatment ameliorates EBI after SAH through the attenuation of oxidative stress and NF-κB-mediated inflammation.

HIG1, a novel regulator of mitochondrial γ-secretase, maintains normal mitochondrial function.

The γ-secretase complex (which contains presenilins, nicastrin, anterior pharynx defective-1, and presenilin enhancer-2) cleaves type I transmembrane proteins, including Notch and amyloid precursor protein. Dysregulated γ-secretase activity has been implicated in the pathogenesis of Alzheimer's disease, stroke, atherosclerosis, and cancer. Tight regulation of γ-secretase activity is required for normal physiology. Here, we isolated HIG1 (hypoxia inducible gene 1, domain member 1A) from a functional screen of γ-secretase inhibitory genes. HIG1 was highly expressed in the brain. Interestingly, HIG1 was localized to the mitochondria and was directly bound to γ-secretase components on the mitochondrial membrane in SK-N-SH neuroblastoma cells. Overexpresssion of HIG1 attenuated hypoxia-induced γ-secretase activation on the mitochondrial membrane and the accumulation of intracellular amyloid ß. This accumulation was accompanied by hypoxia-induced mitochondrial dysfunction. The latter half domain of HIG1 was required for binding to the γ-secretase complex and suppression of γ-secretase activity. Moreover, depletion of HIG1 increased γ-secretase activation and enhanced hypoxia-induced mitochondrial dysfunction. In summary, HIG1 is a novel modulator of the mitochondrial γ-secretase complex, and may play a role in the maintenance of normal mitochondrial function.

Role of central nervous system periostin in cerebral ischemia.

BACKGROUND AND PURPOSE: Although periostin, an extracellular matrix glycoprotein, plays pivotal roles in survival, migration, and regeneration in various cells, its expression and function in the brain are still unknown. Here, we investigated the expression and role of periostin in the ischemic brain. METHODS: Expression of full-length periostin (periostin 1 [Pn1]) and its splicing variant lacking exon 17 (periostin 2 [Pn2]) was examined by real-time reverse transcription polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemical staining in male C57BL6/J mice. The actions of periostin were examined in adult primary neuronal culture and in a transient middle cerebral artery occlusion (tMCAo) model. RESULTS: Expression of Pn2, but not of Pn1, mRNA was markedly changed after tMCAo. Pn2 mRNA was decreased in the ischemic core at 3 hours after ischemia. At 24 hours, Pn2 mRNA was significantly increased in both the peri-ischemic and ischemic regions. Periostin was mainly observed in neurons in normal brain. However, neuronal expression of periostin was decreased temporarily in the ischemic region, but increased in astrocytes and around endothelial cells at 24 hours after tMCAo. Of importance, intracerebroventricular injection of Pn2 resulted in a significant reduction in infarct volume at 24 hours after tMCAo associated with phosphorylation of Akt. Also, the Pn2-treated mice survived longer until 1 week after tMCAo. Pn2 significantly inhibited neuronal death under hypoxia and stimulated neurite outgrowth. CONCLUSIONS: Here, we demonstrated that periostin was expressed in the brain, and exogenous Pn2 exhibited neuroprotective effects and accelerated neurite outgrowth. Additional studies on periostin may provide new insights into the treatment of ischemic stroke.

Perindopril, a centrally active angiotensin-converting enzyme inhibitor, prevents cognitive impairment in mouse models of Alzheimer's disease.

The purpose of this work was to test whether brain-penetrating angiotensin-converting enzyme (ACE) inhibitors (e.g., perindopril), as opposed to non-brain-penetrating ACE inhibitors (e.g., enalapril and imidapril), may reduce the cognitive decline and brain injury in Alzheimer's disease (AD). We first compared the effect of perindopril, enalapril, and imidapril on cognitive impairment and brain injury in a mouse model of AD induced by intracerebroventricular (i.c.v.) injection of amyloid-ß (Aß)1₋40. Perindopril, with significant inhibition of hippocampal ACE, significantly prevented cognitive impairment in this AD mouse model. This beneficial effect was attributed to the suppression of microglia/astrocyte activation and the attenuation of oxidative stress caused by iNOS induction and extracellular superoxide dismutase down-regulation. In contrast, neither enalapril nor imidapril prevented cognitive impairment and brain injury in this AD mouse. We next examined the protective effects of perindopril on cognitive impairment in PS2APP-transgenic mice overexpressing Aß in the brain. Perindopril, without affecting brain Aß deposition, significantly suppressed the increase in hippocampal ACE activity and improved cognition in PS2APP-transgenic mice, being associated with the suppression of hippocampal astrocyte activation and attenuation of superoxide. Our data demonstrated that the brain-penetrating ACE inhibitor perindopril, as compared to non-brain-penetrating ACE inhibitors, protected against cognitive impairment and brain injury in experimental AD models.

Estrogen inhibits vascular calcification via vascular RANKL system: common mechanism of osteoporosis and vascular calcification.

RATIONALE: Arterial calcification and osteoporosis are associated in postmenopausal women. RANK (the receptor activator of nuclear factor kappaB), RANKL (RANK ligand), and osteoprotegerin are key proteins in bone metabolism and have been found at the site of aortic calcification. The role of these proteins in vasculature, as well as the contribution of estrogen to vascular calcification, is poorly understood. OBJECTIVE: To clarify the mechanism of RANKL system to vascular calcification in the context of estrogen deficiency. METHODS AND RESULTS: RANKL induced the calcification inducer bone morphogenetic protein-2 by human aortic endothelial cells (HAECs) and decreased the calcification inhibitor matrix Gla protein (MGP) in human aortic smooth muscle cells (HASMCs), as quantified by real-time PCR and Western blot analysis. RANKL also induced bone-related gene mRNA expression and calcium deposition (Alizarin red staining) followed by the osteogenic differentiation of HASMCs. Estrogen inhibited RANKL signaling in HAECs and HASMCs mainly through estrogen receptor alpha. Apolipoprotein E-deficient mice fed with Western high-fat diet for 3 months presented atherosclerotic calcification (Oil red and Alizarin red staining) and osteoporosis (microcomputed tomographic analysis) after ovariectomy and increased expression of RANKL, RANK, and osteopontin in atherosclerotic lesion, as detected by in situ hybridization. Estrogen replacement inhibited osteoporosis and the bone morphogenetic protein osteogenic pathway in aorta by decreasing phosphorylation of smad-1/5/8 and increasing MGP mRNA expression. CONCLUSIONS: RANKL contributes to vascular calcification by regulating bone morphogenetic protein-2 and MGP expression, as well as bone-related proteins, and is counteracted by estrogen in a receptor-dependent manner.

Blocking Periostin Prevented Development of Inflammation in Rhabdomyolysis-Induced Acute Kidney Injury Mice Model.

BACKGROUND: Rhabdomyolysis is the collapse of damaged skeletal muscle and the leakage of muscle-cell contents, such as electrolytes, myoglobin, and other sarcoplasmic proteins, into the circulation. The glomeruli filtered these products, leading to acute kidney injury (AKI) through several mechanisms, such as intratubular obstruction secondary to protein precipitation. The prognosis is highly mutable and depends on the underlying complications and etiologies. New therapeutic plans to reduce AKI are now needed. Up to now, several cellular pathways, with the nuclear factor kappa beta (NF-kB), as well as the proinflammatory effects on epithelial and tubular epithelial cells, have been recognized as the major pathway for the initiation of the matrix-producing cells in AKI. Recently, it has been mentioned that periostin (POSTN), an extracellular matrix protein, is involved in the development of inflammation through the modulation of the NF-kB pathway. However, how POSTN develops the inflammation protection in AKI by rhabdomyolysis is uncertain. This study aimed to investigate the role of POSTN in a rhabdomyolysis mice model of AKI induced by an intramuscular injection of 50% glycerol. METHODS: In vivo, we performed an intramuscular injection of 50% glycerol (5 mg/kg body weight) to make rhabdomyolysis-induced AKI. We examined the expression level of POSTN through the progression of AKI after glycerol intramuscular injection for C57BL/6J wildtype (WT) mice. We sacrificed mice at 72 h after glycerol injection. We made periostin-null mice to examine the role of POSTN in acute renal failure. The role of periostin was further examined through in vitro methods. The development of renal inflammation is linked with the NF-kB pathway. To examine the POSTN function, we administrated hemin (100 µM) on NIH-3T3 fibroblast cells, and the following signaling pathways were examined. RESULTS: The expression of periostin was highly increased, peaking at about 72 h after glycerol injection. The expression of inflammation-associated mRNAs such as monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-alpha (TNF-a) and IL-6, and tubular injury score in H-E staining were more reduced in POSTN-null mice than WT mice at 72 h after glycerol injection. CONCLUSION: POSTN was highly expressed in the kidney through rhabdomyolysis and was a positive regulator of AKI. Targeting POSTN might propose a new therapeutic strategy against the development of acute renal failure.

Beneficial effects of combination of valsartan and amlodipine on salt-induced brain injury in hypertensive rats.

The optimum antihypertensive treatment for prevention of hypertensive stroke has yet to be elucidated. This study was undertaken to examine the benefit of a combination of valsartan, an angiotensin II type 1 (AT1) receptor blocker, and amlodipine, a calcium channel blocker, in prevention of high-salt-induced brain injury in hypertensive rats. High-salt-loaded stroke-prone spontaneously hypertensive rats (SHRSPs) were given 1) vehicle, 2) valsartan (2 mg/kg/day), 3) amlodipine (2 mg/kg/day), or 4) a combination of valsartan and amlodipine for 4 weeks. The effects on brain injury were compared between all groups. High-salt loading in SHRSPs caused the reduction of cerebral blood flow (CBF), cerebral hypoxia, white matter lesions, glial activation, AT1 receptor up-regulation, endothelial nitric-oxide synthase (eNOS) uncoupling, inducible nitric-oxide synthase induction, and nitroxidative stress. Valsartan, independently of blood pressure, enhanced the protective effects of amlodipine against brain injury, white matter lesions, and glial activation in salt-loaded SHRSPs. These beneficial effects of valsartan added to amlodipine were associated with an additive improvement in CBF and brain hypoxia because of an additive improvement in cerebral arteriolar remodeling and vascular endothelial dysfunction. Furthermore, valsartan added to amlodipine enhanced the attenuation of cerebral nitroxidative stress through an additive suppression of eNOS uncoupling. Valsartan, independently of blood pressure, augmented the protective effects of amlodipine against brain injury in salt-loaded hypertensive rats through an improvement in brain circulation attributed to nitroxidative stress. Our results suggest that the combination of valsartan and amlodipine may be a promising strategy for the prevention of salt-related brain injury in hypertensive patients.

Negative action of hepatocyte growth factor/c-Met system on angiotensin II signaling via ligand-dependent epithelial growth factor receptor degradation mechanism in vascular smooth muscle cells.

RATIONALE: Neointimal hyperplasia contributes to atherosclerosis and restenosis after percutaneous coronary intervention. Vascular injury in each of these conditions results in the release of mitogenic growth factors and hormones that contribute to pathological vascular smooth muscle cell growth and inflammation. Hepatocyte growth factor (HGF) is known as an antiinflammatory growth factor, although it is downregulated in injured tissue. However, the precise mechanism how HGF reduces inflammation is unclear. OBJECTIVE: To elucidate the mechanism how HGF and its receptor c-Met reduces angiotensin II (Ang II)-induced inflammation. METHODS AND RESULTS: HGF reduced Ang II-induced vascular smooth muscle cell growth and inflammation by controlling translocation of SHIP2 (Src homology domain 2-containing inositol 5'-phosphatase 2), which led to Ang II-dependent degradation of epithelial growth factor receptor. Moreover, the present study also revealed a preventive effect of HGF on atherosclerotic change in an Ang II infusion and cuff HGF transgenic mouse model. CONCLUSIONS: These data suggest that the HGF/c-Met system might regulate extrinsic factor signaling that maintains the homeostasis of organs.

The ATP-binding cassette transporter BCRP1/ABCG2 plays a pivotal role in cardiac repair after myocardial infarction via modulation of microvascular endothelial cell survival and function.

OBJECTIVE: To clarify the impact of breast cancer resistance protein 1 (BCRP1)/ATP-binding cassette transporter subfamily G member 2 (ABCG2) expression on cardiac repair after myocardial infarction (MI). METHODS AND RESULTS: The ATP-binding cassette transporter BCRP1/ABCG2 is expressed in various organs, including the heart, and may regulate several tissue defense mechanisms. BCRP1/ABCG2 was mainly expressed in endothelial cells of microvessels in the heart. MI was induced in 8- to 12-week-old wild-type (WT) and Bcrp1/Abcg2 knockout (KO) mice by ligating the left anterior descending artery. At 28 days after MI, the survival rate was significantly lower in KO mice than in WT mice because of cardiac rupture. Echocardiographic, hemodynamic, and histological assessments showed that ventricular remodeling was more deteriorated in KO than in WT mice. Capillary, myofibroblast, and macrophage densities in the peri-infarction area at 5 days after MI were significantly reduced in KO compared with WT mice. In vitro experiments demonstrated that inhibition of BCRP1/ABCG2 resulted in accumulation of intracellular protoporphyrin IX and impaired survival of microvascular endothelial cells under oxidative stress. Moreover, BCRP1/ABCG2 inhibition impaired migration and tube formation of endothelial cells. CONCLUSIONS: BCRP1/ABCG2 plays a pivotal role in cardiac repair after MI via modulation of microvascular endothelial cell survival and function.

Periostin Short Fragment with Exon 17 via Aberrant Alternative Splicing Is Required for Breast Cancer Growth and Metastasis.

BACKGROUND: Periostin (POSTN) is a 93 kDa matrix protein that helps to regulate collagen gene expression in the extracellular matrix. POSTN overexpression is a prognostic factor in malignant cancers; however, some researchers have observed it in the stroma, whereas others have reported it on tumors. OBJECTIVE: This study aimed to investigate the function of POSTN on tumors. METHODS AND RESULTS: We found that POSTN in cancer cells can be detected by using an antibody against the POSTN C-terminal region exon 17 (Ex17 antibody), but not with an antibody against the POSTN N-terminal region exon 12 (Ex12 antibody) in patients with breast cancer. In a fraction secreted from fibroblasts, LC-MS/MS analysis revealed a short fragment of POSTN of approximately 40 kDa with exon 17. In addition, molecular interaction analysis showed that POSTN with exon 17, but not POSTN without exon 17, bound specifically to wnt3a, and the Ex17 antibody inhibited the binding. CONCLUSION: A short fragment of POSTN with exon 17, which originates in the fibroblasts, is transported to cancer cells, whereas POSTN fragments without exon 17 are retained in the stroma. The Ex17 antibody inhibits the binding between POSTN exon 17 and wnt3a.

Periostin Exon-21 Antibody Neutralization of Triple-Negative Breast Cancer Cell-Derived Periostin Regulates Tumor-Associated Macrophage Polarization and Angiogenesis.

Periostin (Pn) is involved in multiple processes of cancer progression. Previously, we reported that Pn expression is correlated with mesenchymal tumor markers and poor prognosis in triple-negative breast cancer (TNBC). In the TNBC xenograft model, chemotherapy increased expression of a Pn alternative splicing variant (ASV) with exon 21, and administration of the neutralizing antibody against Pn with exon 21 (Pn-21 Ab) overcame chemoresistance with a reduction in the mesenchymal cancer cell fraction. In the present study, the role of Pn ASV with exon 21 in TNBC progression has been addressed. We first established a stable cell line carrying a fluorescence-based splicing reporter. Pn-positive TNBC has higher expression of genes related to tumor-associated macrophage (TAM) recruitment and ECM-receptor interaction than Pn-negative cells. In a xenograft model, only Pn-positive cells initiated tumor formation, and the Pn-21 Ab suppressed tumor cell growth, accompanied by decreased M2 TAM polarization and the number of tumor vessels. These data suggest that cancer cell-derived Pn ASV educates TAMs and regulates angiogenesis, which in turn establishes a microenvironmental niche that is supportive of TNBC.

CHF1/Hey2 plays a pivotal role in left ventricular maturation through suppression of ectopic atrial gene expression.

We previously reported that mice lacking the hairy-related basic helix-loop-helix (bHLH) transcription factor CHF1/Hey2 develop a thin-walled left ventricle. To explore the basis for this phenotype, we examined regional gene expression patterns in the developing myocardium. We found that atrial natriuretic factor (ANF), which is normally expressed in the atria and trabeculae and is restricted from the developing compact myocardium beginning at embryonic day 13.5, is persistently expressed in the left ventricular compact myocardium of the knockout animals. We also examined the expression pattern of the T-box transcription factor Tbx5, a known regulator of ANF, and an additional Tbx5-dependent gene, connexin 40 (Cx40), both of which share a similar expression pattern to ANF during development. Tbx5 and Cx40 were similarly expressed ectopically in the compact myocardium of the CHF1/Hey2 knockout mouse. The atrial contractile genes mlc1a and mlc2a were also expressed ectopically in the left ventricular compact myocardium, providing evidence for a general dysregulation of atrial gene expression. Crossing of a myocardial-specific CHF1/Hey2 transgenic mouse with the knockouts led to rescue of the thin-walled myocardial phenotype and restoration of the normal patterns of gene expression. Myocardial cell proliferation, which has been shown previously to be suppressed by Tbx5, was also decreased in the knockout mice and rescued by the transgene. Our findings suggest that CHF1/Hey2 suppresses atrial identity in the left ventricular compact myocardium, facilitates myocardial proliferation by suppressing Tbx5, and thereby promotes proper ventricular myocardial maturation.

Candesartan ameliorates cardiac dysfunction observed in angiotensin-converting enzyme 2-deficient mice.

The renin-angiotensin (Ang) system plays a critical role in the regulation of blood pressure, body fluid, electrolyte homeostasis, and organ remodeling under physiological and pathological conditions. The carboxypeptidase ACE2 is a homologue of angiotensin-converting enzyme (ACE). It has been reported that ACE2-deficient mice develop cardiac dysfunction with increased plasma levels of Ang II. However, the molecular mechanism by which genetic disruption of ACE2 results in heart dysfunction is not fully understood. Here, we generated mice with targeted disruption of the Ace2 gene and compared the cardiovascular function of ACE2(-/y) mice with that of their wild-type littermates. ACE2-deficient mice were viable and fertile and lacked any gross structural abnormalities. Echocardiographic study detected no functional difference between ACE2(-/y) and wild-type mice at 12 weeks of age. Twenty-four-week-old ACE2(-/y) mice displayed significantly enlarged hearts with impaired systolic and diastolic function. The Ang II level was elevated in the plasma and heart of ACE2(-/y) mice. Pharmacological blockade of Ang II type 1 receptor (AT1) with candesartan attenuated the development of cardiac dysfunction in ACE2(-/y) mice. These results suggest that enhanced stimulation of AT1 may play a role in the development of cardiac dysfunction observed in ACE2-deficient mice.

A model of cerebrovascular injury in rats.

Although the pathophysiology of post-angioplasty restenosis has been extensively studied in extracranial arteries using transluminal vascular injury model in rodents, it is still not well known in the intracranial arteries, which have quite different structures from extracranial arteries. Here, we examined whether 1-min placement of modified intraluminal suture could induce an injury in the internal carotid artery (ICA) in rats and observed temporal profile of histological change after the injury. HE staining showed that the injured intracranial ICA was dilated, while the media was markedly thinned at 1 day after injury. The internal elastic lamina was not observed, and the media contained few cells. At 1 week after injury, a thin layer of neointimal hyperplasia was observed on the luminal side of the internal elastic lamina. Neointimal hyperplasia developed until at least 4 weeks after injury. Morphometric analysis demonstrated that the healing process of the injury was related to arterial remodeling. Immunohistochemical staining for alpha-smooth muscle actin and electron microscopic analysis showed that the neointima was composed of smooth muscle cells. Re-endothelialization was observed from 1 to 4 weeks after injury by immunohistochemical staining for von Willebrand's factor and electron microscopic analysis. Vascular endothelial growth factor was expressed in neointima on days 7 and 14. Interestingly, superoxide anion was not increased in injured arteries on day 3, when the infiltration of macrophages was intensive, but increased on day 7, when infiltrating macrophages almost disappeared. These findings might shed new light on pathophysiology of post-angioplasty restenosis in intracranial arteries.