Effect of basal insulin therapy on vascular endothelial function and adipokine profiles in people with Type 2 diabetes.

AIM: To compare the effects of the basal insulin analogues glargine and detemir on endothelial function and adipocytokine levels in people with Type 2 diabetes. METHODS: We studied 32 people with Type 2 diabetes whose blood glucose control was unsatisfactory while receiving only oral hypoglycaemic drugs. Participants were randomized to either insulin glargine or detemir for 24 weeks and then crossed over to the other treatment without a washout period. Flow-mediated vasodilatation, adipocytokine levels (plasminogen activator inhibitor-1 and leptin/adiponectin ratio), and fasting ghrelin levels were monitored. RESULTS: HbA1c levels were significantly decreased by both basal insulin therapies. Body weight was significantly increased by glargine but not by detemir. The proportion of flow-mediated vasodilatation was significantly increased by detemir but not glargine (glargine: from 5.17 ± 0.69 to 5.94 ± 0.83%; detemir: from 4.89 ± 0.78 to 7.92 ± 0.69%). Plasminogen activator inhibitor-1 level was significantly decreased by only detemir (glargine: from 16.4 ± 1.8 to 17.3 ± 2.1; detemir: from 19.2 ± 2.8 to 16.0 ± 1.6 ng/ml). The leptin/adiponectin ratio was significantly increased only by glargine. Acyl ghrelin level was significantly decreased by glargine but not detemir. CONCLUSIONS: These results suggest that the effect on endothelial function and adipocytokine profiles may differ between glargine and detemir in people with diabetes (Trial registration ID: UMIN000004973).

Impact of B-type natriuretic peptide (BNP) on development of atrial fibrillation in people with Type 2 diabetes.

AIMS: To examine if a simple biomarker can identify people with diabetes who are at high risk of atrial fibrillation. METHODS: A retrospective cohort study was conducted at a single centre in people with Type 2 diabetes referred to our department between January 2000 and December 2007. In 517 consecutive people without any history, signs or symptoms of atrial fibrillation at baseline, the association between baseline B-type natriuretic peptide level and future atrial fibrillation incidence was examined, with adjustments for other potentially confounding factors. RESULTS: A total of 28 people were diagnosed with new-onset atrial fibrillation during a median 6-year follow-up. When people were categorized into three groups according to B-type natriuretic peptide clinical thresholds (20 and 100 pg/ml), hazard ratios for the development of atrial fibrillation in the middle and highest B-type natriuretic peptide groups were 2.8 and 9.4, respectively, compared with the lowest B-type natriuretic peptide group. Time-dependent receiver-operating curve analysis identified a threshold for B-type natriuretic peptide to detect atrial fibrillation development of 52.8 pg/ml (sensitivity 75.2%, specificity 68.8%). The B-type natriuretic peptide predictive value was independent of and similar to that of left atrial size and ventricular dimension. CONCLUSION: In people with Type 2 diabetes, high baseline B-type natriuretic peptide levels were significantly associated with future atrial fibrillation development.

Germline deletion and a somatic mutation of the PRKAR1A gene in a Carney complex-related pituitary adenoma.

OBJECTIVE: The objective was to assess involvement of loss of the PRKAR1A gene encoding a type 1α regulatory subunit of cAMP-dependent protein kinase A located on 17q24 in a Carney complex (CNC)-related pituitary adenoma. DESIGN: We investigated aberrations of the PRKAR1A gene in a CNC patient with a GH-producing pituitary adenoma, whose family has three other members with probable CNC. METHODS: A gene mutation was identified by a standard DNA sequencing method based on PCR. DNA copy number was measured to evaluate allelic loss on 17q24 by quantitative PCR. The breakpoints of deletion were determined by cloning a rearranged region in the deleted allele. RESULTS: A PRKAR1A mutation of c.751_758del8 (p.S251LfsX16) was found in genomic DNA obtained from a pituitary adenoma, but not leukocytes from the patient. Reduced DNA copy number at loci including the PRKAR1A gene on 17q24 was detected in both the tumor and leukocytes, suggesting a deletion at the loci at the germline level. The deletion size was determined to be ∼ 0.5 Mb and this large deletion was also found in two other family members. CONCLUSION: This is the first case showing a CNC-related pituitary adenoma with the combination of somatic mutation and a large inherited deletion of the PRKAR1A gene. Biallelic inactivation of PRKAR1A appears to be necessary for the development of CNC-related pituitary adenoma.

Induced pluripotent stem cells generated from diabetic patients with mitochondrial DNA A3243G mutation.

AIMS/HYPOTHESIS: The aim of this study was to generate induced pluripotent stem (iPS) cells from patients with mitochondrial DNA (mtDNA) mutation. METHODS: Skin biopsies were obtained from two diabetic patients with mtDNA A3243G mutation. The fibroblasts thus obtained were infected with retroviruses encoding OCT4 (also known as POU5F1), SOX2, c-MYC (also known as MYC) and KLF4. The stem cell characteristics were investigated and the mtDNA mutation frequencies evaluated by Invader assay. RESULTS: From the two diabetic patients we isolated four and ten putative mitochondrial disease-specific iPS (Mt-iPS) clones, respectively. Mt-iPS cells were cytogenetically normal and positive for alkaline phosphatase activity, with the pluripotent stem cell markers being detectable by immunocytochemistry. The cytosine guanine dinucleotide islands in the promoter regions of OCT4 and NANOG were highly unmethylated, indicating epigenetic reprogramming to pluripotency. Mt-iPS clones were able to differentiate into derivatives of all three germ layers in vitro and in vivo. The Mt-iPS cells exhibited a bimodal degree of mutation heteroplasmy. The mutation frequencies decreased to an undetectable level in six of 14 clones, while the others showed several-fold increases in mutation frequencies (51-87%) compared with those in the original fibroblasts (18-24%). During serial cell culture passage and after differentiation, no recurrence of the mutation or no significant changes in the levels of heteroplasmy were seen. CONCLUSIONS/INTERPRETATION: iPS cells were successfully generated from patients with the mtDNA A3243G mutation. Mutation-rich, stable Mt-iPS cells may be a suitable source of cells for human mitochondrial disease modelling in vitro. Mutation-free iPS cells could provide an unlimited, disease-free supply of cells for autologous transplantation therapy.

Vasodilator therapy with hydralazine induces angiotensin AT receptor-mediated cardiomyocyte growth in mice lacking guanylyl cyclase-A.

BACKGROUND AND PURPOSE: Recent clinical guidelines advocate the use of the isosorbide dinitrate/hydralazine combination in treatment for heart failure. However, clinical and laboratory evidence suggest that some vasodilators may induce cardiac hypertrophy under uncertain conditions. This study investigated the effects and underlying mechanism of action of the vasodilator hydralazine on cardiac growth. EXPERIMENTAL APPROACH: Wild-type mice and animals deficient in guanylyl cyclase-A (GCA) and/or angiotensin receptors (AT(1) and AT(2) subtypes) were treated with hydralazine ( approximately 24 mg.kg(-1).day(-1) in drinking water) for 5 weeks. Cardiac mass and/or cardiomyocyte cross-sectional area, fibrosis (van Giessen-staining) and cardiac gene expression (real-time RT-PCR) were measured. KEY RESULTS: Hydralazine lowered blood pressure in mice of all genotypes. However, this treatment increased the heart and left ventricular to body weight ratios, as well as cardiomyocyte cross-sectional area, and cardiac expression of atrial natriuretic peptide mRNA in mice lacking GCA. Hydralazine did not affect cardiac hypertrophy in wild-type mice and mice lacking either AT(1) or AT(2) receptors alone. However, the pro-hypertrophic effect of hydralazine was prevented in mice lacking both GCA and AT(2), but not GCA and AT(1) receptors. However, hydralazine did decrease cardiac collagen deposition and collagen I mRNA (signs of cardiac fibrosis) in mice that were deficient in GCA, or both GCA and AT(2) receptors. CONCLUSIONS AND IMPLICATIONS: The vasodilator hydralazine induced AT(2) receptor-mediated cardiomyocyte growth under conditions of GCA deficiency. However, attenuation of cardiac fibrosis by hydralazine could be beneficial in the management of cardiac diseases.

Decreased circulating CD34+ cells are associated with progression of diabetic nephropathy.

AIMS: Circulating progenitor cells such as CD34+ cells play a key role in maintenance of vascular endothelial function and neovascularization, and a decrease in the number of CD34+ cells is associated with cardiovascular disease. However, the contribution of circulating progenitor cells to microvascular disease, such as diabetic nephropathy, is unclear. This study was therefore designed to clarify the association between diabetic nephropathy and circulating CD34+ cells. METHODS: We measured circulating CD34+ cell numbers in 85 Type 2 diabetic patients aged 40-70 years with normo- and microalbuminuria and determined the association with urinary albumin excretion rate (UAER). RESULTS: The number of circulating CD34+ cells significantly correlated with log UAER (r = -0.289, P = 0.008). Furthermore, in patients with low numbers of CD34+ cells (0.68 > cells/microl, lowest quartile of CD34+ cell number) UAER increased significantly after 12 months compared with baseline [from 34.3 +/- 7.0 to 53.6 +/- 10.8 mg/g creatinine (gCr), P < 0.05], whereas in patients with a high number of CD34+ cells (1.0 < cells/microl, highest quartile of CD34+ cell number) UAER did not change (from 16.7 +/- 4.8 to 20.1 +/- 3.0 mg/gCr). CONCLUSIONS: These results suggest that a decreased number of circulating CD34+ cells is involved in the progression of diabetic nephropathy and may be a predictor of the disease.

C-type natriuretic peptide is a Schwann cell-derived factor for development and function of sensory neurones.

Cyclic GMP (cGMP) is known to play important roles for neuronal development and neurite pathfinding. However, the regulatory mechanism that governs the synthesis of cGMP in the nervous system is not well defined. In the present study, we examined the role of C-type natriuretic peptide (CNP), which increases intracellular cGMP upon binding to its receptor, guanylyl cyclase (GC)-B, in the peripheral nervous system. Immunohistochemistry revealed that CNP is demonstrated in Schwann cells, whereas GC-B mRNA is highly expressed in dorsal root ganglion (DRG) neurones. In cultured DRG neurones, GC-B was demonstrated in dendrites of TrkA-positive cells, where it co-exists with cGMP-dependent protein kinase I (cGKI), the major intracellular mediator of cGMP actions. Addition of CNP in the culture medium increased the density of fine neurites, which was accompanied by the increase in phosphorylation of vasodilator-stimulated phosphoprotein, a cGKI substrate. Furthermore, in mice deficient for the CNP gene (CNP-KO), the numbers of TrkA-positive DRG neurones were diminished. Likewise, there were much less cGKI-positive neurones in DRG and cGKI-positive fibres in the dorsal spinal cord of CNP-KO than wild-type mice. Finally, the bone deformity-rescued CNP-KO mice displayed a decreased response to formalin-induced pain compared to wild-type. Taken together, these results suggest that CNP is derived from Schwann cells and plays an important role for the development and function of nociceptive sensory neurones.

Tumour budding at invasive margins and outcome in colorectal cancer.

OBJECTIVE: Tumour budding, defined as small clusters of undifferentiated cancer cells at invasive margins, has been shown to reflect biologic aggressiveness of colorectal cancers. We therefore examined the prognostic significance of tumour budding in patients with colorectal carcinoma, particularly focusing on comparisons with other clinicopathological findings. METHOD: Tumour budding was investigated in surgically resected specimens from 159 patients with colorectal carcinoma. With haematoxylin and eosin stained slides containing the entire invasive margin, the degree of tumour budding was classified into three grades: mild, <1/3 of the entire invasive margin; moderate, 1/3-2/3; marked, >2/3. RESULTS: Mild tumour budding was found in 54 (34%) cases, moderate in 59 (37%) cases and marked in 46 (29%) cases. The degree of budding was linked with poor tumour differentiation, lymph node metastasis and advanced TNM stage (P < 0.001). In univariate analysis, patients with marked tumour budding [5-year cancer-related survival (CRS)/recurrence-free survival (RFS), 39%/53%] had significantly worse survival [CRS, hazard ratio (HR), 4.561; 95% confidence interval (CI), 2.265-9.184; P < 0.001; RFS, HR, 3.240; 95% CI, 1.430-7.342; P = 0.005] than those with mild (5-year CRS/RFS, 80%/82%) or moderate (63%/66%) budding. In the Cox regression model, marked tumour budding (HR, 3.137; 95% CI, 1.517-6.487; P = 0.002) and advanced tumour stage (stage III, HR, 3.226; 95% CI, 1.475-7.053; P = 0.003; stage IV, HR, 24.443; 95% CI, 10.843-55.100; P < 0.001) proved to be an independent predictor of short CRS. CONCLUSION: Tumour budding is a practical and significant histological index for identification of high malignant potential and poor outcome in patients with colorectal carcinoma.

SIRT2, a tubulin deacetylase, acts to block the entry to chromosome condensation in response to mitotic stress.

We previously identified SIRT2, an nicotinamide adenine dinucleotide (NAD)-dependent tubulin deacetylase, as a protein downregulated in gliomas and glioma cell lines, which are characterized by aneuploidy. Other studies reported SIRT2 to be involved in mitotic progression in the normal cell cycle. We herein investigated whether SIRT2 functions in the mitotic checkpoint in response to mitotic stress caused by microtubule poisons. By monitoring chromosome condensation, the exogenously expressed SIRT2 was found to block the entry to chromosome condensation and subsequent hyperploid cell formation in glioma cell lines with a persistence of the cyclin B/cdc2 activity in response to mitotic stress. SIRT2 is thus a novel mitotic checkpoint protein that functions in the early metaphase to prevent chromosomal instability (CIN), characteristics previously reported for the CHFR protein. We further found that histone deacetylation, but not the aberrant DNA methylation of SIRT2 5'untranslated region is involved in the downregulation of SIRT2. Although SIRT2 is normally exclusively located in the cytoplasm, the rapid accumulation of SIRT2 in the nucleus was observed after treatment with a nuclear export inhibitor, leptomycin B and ionizing radiation in normal human fibroblasts, suggesting that nucleo-cytoplasmic shuttling regulates the SIRT2 function. Collectively, our results suggest that the further study of SIRT2 may thus provide new insights into the relationships among CIN, epigenetic regulation and tumorigenesis.

Apparent B-type natriuretic peptide selectivity in the kidney due to differential processing.

Two natriuretic peptides, atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), are found principally in the heart. In preliminary experiments with mouse kidney cells or slices, we found mouse BNP1-45 much more potent than ANP1-28 in causing elevations of cGMP (>50-fold). The guanylyl cyclase-A (GC-A) receptor has been suggested to represent the primary means by which both peptides signal. In cultured cells overexpressing GC-A, BNP and ANP were almost equivalent in potency, suggesting that a receptor unique for BNP exists in the kidney. However, in mice lacking the GC-A gene, neither BNP nor ANP significantly elevated cGMP in kidney slices. Phosphoramidon, a neutral endopeptidase inhibitor, shifted the apparent potency of ANP to values equivalent to that of BNP, suggesting these kidney cell/slices rapidly degrade ANP but not BNP. Mass spectroscopic analysis confirmed that ANP is rapidly cleaved at the first cysteine of the disulfide ring, whereas BNP is particularly stable to such cleavage. Other tissues (heart, aorta) failed to significantly degrade ANP or BNP, and therefore the kidney-specific degradation of ANP provides a mechanism for preferential regulation of kidney function by BNP independent of peripheral ANP concentration.

Blockade of the natriuretic peptide receptor guanylyl cyclase-A inhibits NF-kappaB activation and alleviates myocardial ischemia/reperfusion injury.

Acute myocardial infarction (AMI) remains the leading cause of death in developed countries. Although reperfusion of coronary arteries reduces mortality, it is associated with tissue injury. Endothelial P-selectin-mediated infiltration of neutrophils plays a key role in reperfusion injury. However, the mechanism of the P-selectin induction is not known. Here we show that infarct size after ischemia/reperfusion was significantly smaller in mice lacking guanylyl cyclase-A (GC-A), a natriuretic peptide receptor. The decrease was accompanied by decreases in neutrophil infiltration in coronary endothelial P-selectin expression. Pretreatment with HS-142-1, a GC-A antagonist, also decreased infarct size and P-selectin induction in wild-type mice. In cultured endothelial cells, activation of GC-A augmented H2O2-induced P-selectin expression. Furthermore, ischemia/reperfusion-induced activation of NF-kappaB, a transcription factor that is known to promote P-selectin expression, is suppressed in GC-A-deficient mice. These results suggest that inhibition of GC-A alleviates ischemia/reperfusion injury through suppression of NF-kappaB-mediated P-selectin induction. This novel, GC-A-mediated mechanism of ischemia/reperfusion injury may provide the basis for applying GC-A blockade in the clinical treatment of reperfusion injury.

Induction of JAB/SOCS-1/SSI-1 and CIS3/SOCS-3/SSI-3 is involved in gp130 resistance in cardiovascular system in rat treated with cardiotrophin-1 in vivo.

CIS (cytokine-inducible SH2 protein), SOCS (suppressor of cytokine signaling), or SSI (signal transducers and activators of transcription [STAT]-induced STAT inhibitor) proteins are a family of cytokine-inducible negative regulators of cytokine signaling via Janus kinase (JAK)-STAT pathways. Given the evidence that the JAK-STAT pathway plays a critical role in the cardiovascular system, the primary objective of this study was to assess the effects of the CIS family on JAK-STAT signaling in the cardiovascular system in rats treated with cardiotrophin-1 (CT-1), an interleukin-6 family of cytokines. Intravenous injection of 20 microgram/kg body weight of CT-1 induced a transient, marked increase in STAT3 activation in various tissues, including heart and lung, and subsequent upregulation of 2 members of the CIS family, JAK-binding protein (JAB)/SOCS-1/SSI-1 and CIS3/SOCS-3/SSI-3, in the same tissues. It was also observed that CIS3 was directly associated with JAK2 in vivo. Pretreatment with the same dose of CT-1 60 minutes before significantly attenuated the STAT3 activation induced by a second injection of CT-1. We previously reported that intravenous injection of CT-1 results in the nitric oxide (NO)-dependent hypotension accompanied by the induction of inducible NO synthase mRNA. In rats pretreated with CT-1, the induction of inducible NO synthase mRNA or hypotension by subsequent CT-1 injection was not observed. Forced expression of JAB or CIS3, but not other CISs, directly blocked CT-1-induced STAT3 activation in 293 cells. These results suggest that JAB and CIS3 serve as endogenous inhibitors of CT-1-mediated JAK-STAT signaling in the cardiovascular system in vivo.

The neuron-restrictive silencer element-neuron-restrictive silencer factor system regulates basal and endothelin 1-inducible atrial natriuretic peptide gene expression in ventricular myocytes.

Induction of the atrial natriuretic peptide (ANP) gene is a common feature of ventricular hypertrophy. A number of cis-acting enhancer elements for several transcriptional activators have been shown to play central roles in the regulation of ANP gene expression, but much less is known about contributions made by transcriptional repressors. The neuron-restrictive silencer element (NRSE), also known as repressor element 1, mediates repression of neuronal gene expression in nonneuronal cells. We found that NRSE, which is located in the 3' untranslated region of the ANP gene, mediated repression of ANP promoter activity in ventricular myocytes and was also involved in the endothelin 1-induced increase in ANP gene transcription. The repression was conferred by a repressor protein, neuron-restrictive silencer factor (NRSF). NRSF associated with the transcriptional corepressor mSin3 and formed a complex with histone deacetylase (HDAC) in ventricular myocytes. Trichostatin A (TSA), a specific HDAC inhibitor, relieved NRSE-mediated repression of ANP promoter activity, and chromatin immunoprecipitation assays revealed the involvement of histone deacetylation in NRSE-mediated repression of ANP gene expression. Furthermore, in myocytes infected with recombinant adenovirus expressing a dominant-negative form of NRSF, the basal level of endogenous ANP gene expression was increased and a TSA-induced increase in ANP gene expression was apparently attenuated, compared with those in myocytes infected with control adenovirus. Our findings show that an NRSE-NRSF system plays a key role in the regulation of ANP gene expression by HDAC in ventricular myocytes and provide a new insight into the role of the NRSE-NRSF system outside the nervous system.

A genetic model provides evidence that the receptor for atrial natriuretic peptide (guanylyl cyclase-A) inhibits cardiac ventricular myocyte hypertrophy.

Guanylyl cyclase-A (NPR-A; GC-A) is the major and possibly the only receptor for atrial natriuretic peptide (ANP) or B-type natriuretic peptide. Although mice deficient in GC-A display an elevated blood pressure, the resultant cardiac hypertrophy is much greater than in other mouse models of hypertension. Here we overproduce GC-A in the cardiac myocytes of wild-type or GC-A null animals. Introduction of the GC-A transgene did not alter blood pressure or heart rate as a function of genotype. Cardiac myocyte size was larger (approximately 20%) in GC-A null than in wild-type animals. However, introduction of the GC-A transgene reduced cardiac myocyte size in both wild-type and null mice. Coincident with the reduction in myocyte size, both ANP mRNA and ANP content were significantly reduced by overexpression of GC-A, and this reduction was independent of genotype. This genetic model, therefore, separates a regulation of cardiac myocyte size by blood pressure from local regulation by a GC-mediated pathway.

The heart is a source of circulating cardiotrophin-1 in humans.

Cardiotrophin-1 (CT-1) is a new member of the interleukin (IL)-6 family of cytokines and one of the endogenous ligands for gp130 signaling pathways in the heart, which has potent hypertrophic and survival effects on cardiac myocytes. However, the clinical significance of CT-1 is poorly understood, mainly because there is no widely applicable specific and sensitive assay system for measuring plasma levels of circulating CT-1. We therefore developed a competitive radioimmunoassay (RIA) for human CT-1 with rabbit antiserum recognizing the N-terminus region of human CT-1 and using recombinant human CT-1 as a calibrator. The assay displays no cross-reactivities with any of the IL-6 family of cytokines including IL-11, leukemia inhibitory factor, ciliary neurotrophic factor, and oncostatin M. The lower detection limit in buffer was found to be 43 fmol/ml, and the working range was 120-8300 fmol/ml (CV < 15%). This RIA directly recognizes CT-1-like immunoreactivity in human plasma with a mean value of 571 +/- 75 fmol/ml (mean +/- SD) in healthy volunteers. The RIA coupled with gel filtration chromatographic analyses showed that the major molecular form of circulating CT-1 corresponds to recombinant full-length human CT-1. Moreover, there is a significant increase in the plasma CT-1 concentration from the aorta and coronary sinus, which clearly indicates that the heart secretes CT-1 via the coronary sinus into the peripheral circulation. This RIA should serve as a powerful tool for investigating the clinical significance of CT-1.

Lack of association between T-786-->C mutation in the 5'-flanking region of the endothelial nitric oxide synthase gene and essential hypertension.

Accumulating evidence strongly suggests that an alteration in nitric oxide metabolism is involved in the pathogenesis of hypertension. We recently found 2 polymorphisms in the endothelial nitric oxide synthase (eNOS) gene, a Glu298Asp missense variant in exon 7 and a T-786-->C variant in the 5'-flanking region, which are not linked to each other. In our previous reports, we showed a positive association between the Glu298Asp variant and essential hypertension, myocardial infarction, and coronary spastic angina. We also revealed that the T-786-->C variant is strongly associated with coronary spastic angina and leads to the reduction of the eNOS gene promoter activity. To further investigate the genetic involvement of the eNOS gene in essential hypertension, we examined the frequency of T-786-->C variant in two independent populations of persons with essential hypertension in Kyoto (n=215) and Kumamoto (n=186) and compared the frequency with that in each age- and gender-matched control (233 controls in Kyoto and 223 controls in Kumamoto). In both groups, the frequency of T-786-->C variant was similar in patients with hypertension and normal controls. In conclusion, the T-786-->C variant was not positively associated with essential hypertension. Given the evidence of positive association of another polymorphism in the eNOS gene, the Glu298Asp polymorphism, with essential hypertension, special attention will be required to interpret the results of a case-control study for genetic risk factors.

Replication protein A1 reduces transcription of the endothelial nitric oxide synthase gene containing a -786T-->C mutation associated with coronary spastic angina.

We recently reported that a mutation (-786T-->C) in the promoter region of the endothelial nitric oxide synthase (eNOS) gene reduced transcription of the gene and was strongly associated with coronary spastic angina and myocardial infarction. To elucidate the molecular mechanism for the reduced eNOS gene transcription, we have now purified a protein that specifically binds to the mutant allele in nuclear extracts from HeLa cells. The purified protein was identical to replication protein A1 (RPA1), known as a single-stranded DNA binding protein essential for DNA repair, replication and recombination. In human umbilical vein endothelial cells, inhibition of RPA1 expression using antisense oligonucleotide restored transcription driven by the mutated promoter sequence, whereas, conversely, overexpression of RPA1 further reduced it. RPA1 was similarly detected in placenta and eNOS mRNA levels in placentas carrying the -786T-->C mutation were significantly lower than in placentas without it. The functional importance of the diminished eNOS expression was revealed by the finding that serum nitrite/nitrate levels among individuals carrying the -786T-->C mutation were significantly lower than among those without the mutation. RPA1 thus apparently functions as a repressor protein in the -786T-->C mutation-related reduction of eNOS gene transcription associated with the development of coronary artery disease.

Genetic models reveal that brain natriuretic peptide can signal through different tissue-specific receptor-mediated pathways.

Brain natriuretic peptide (BNP), a hormone produced primarily by the cardiac ventricle, is thought to be involved in a variety of homeostatic processes through its cognate receptor, guanylyl cyclase A (GC-A). We previously created transgenic mice overexpressing BNP under the control of the liver-specific human serum amyloid P component promoter (BNP-transgenic mice) and demonstrated that they exhibit reduced blood pressure and cardiac weight accompanied by an elevation of plasma cGMP concentrations and marked skeletal overgrowth through the activation of endochondral ossification. To address whether BNP exerts its biological effects solely through GC-A, we produced BNP-transgenic mice lacking GC-A (BNP-Tg/GC-A-/- mice) and examined their cardiovascular and skeletal phenotypes. The GC-A-/- mice are hypertensive with cardiac hypertrophyrelative to wild-type littermates, which is not alleviated by overexpression of BNP in BNP-Tg/GC-A-/- mice. The BNP-Tg/GC-A-/- mice, however, continue to exhibit marked longitudinal growth of vertebrae and long bones comparably to BNP-Tg mice. This study provides genetic evidence that BNP reduces blood pressure and cardiac weight through GC-A, whereas it dramatically alters endochondral ossification in the absence of this receptor. Therefore, the BNP-Tg/GC-A-/- mice provide the first experimental model demonstrating that this natriuretic peptide can signal in a tissue-specific manner through a receptor other than GC-A.

Cardiotrophin-1 phosphorylates akt and BAD, and prolongs cell survival via a PI3K-dependent pathway in cardiac myocytes.

Growth factors and cytokines trigger survival signaling in a wide variety of cell systems, including cardiac myocytes. Participation of the phosphatidylinositol 3-OH kinase (PI3K)/Akt pathway in survival signaling has already been described in some cell types, but its involvement in the survival of cardiac myocytes is as yet unknown. Recently, CT-1, an interleukin 6-related cytokine, was shown to have survival-promoting, anti-apoptotic effects on cultured cardiac myocytes. However, roles of PI3K-dependent pathways in this signaling have not been elucidated. In the present study, therefore, we examined the participation of the PI3K/Akt pathway in CT-1-induced, survival-promoting signaling in cultured ventricular myocytes. It was found that CT-1 phosphorylated and activated Akt, and the effect was blocked by the PI3K inhibitors LY294002 and wortmannin. CT-1 also phosphorylated the pro-apoptotic factor, BAD, and the BAD phosphorylation was inhibited by LY294002, suggesting that phosphorylation of BAD is one of the key events by which the PI3K/Akt pathway mediates CT-1-induced survival signaling. Further, CT-1 PI3K-dependently prolonged the survival of serum-starved ventricular myocytes by preventing apoptosis. In summary, our findings show that PI3K-dependent survival signals contribute to CT-1-mediated ventricular myocyte survival. In vivo, the death of ventricular myocytes leads to heart failure, and downregulation of survival signals and/or augmentation of pro-apoptotic signals are likely to be important components of disease processes. Thus, the extent to which CT-1 and the PI3K/Akt pathway mitigate such pathological processes, in vivo, is an important question for the future.