Exercise-induced alterations of retinal vessel diameters and cardiovascular risk reduction in obesity.

BACKGROUND: The retinal microcirculation is affected early in the process of atherosclerosis and retinal vessel caliber is an emerging cardiovascular risk factor. Obesity is associated with vascular dysfunction. Here, we investigate the effect of regular exercise on retinal vessel diameters in lean and obese runners. We analyze a possible link to alterations of the nitric oxide (NO)-asymmetric dimethylarginine (ADMA) pathway. METHODS: Retinal vessel diameters were assessed by means of a static vessel analyzer (SVA-T) in 15 obese athletes (OA), 14 lean amateur athletes (AA) and 17 lean elite athletes (EA) following a 10 week training program. ADMA serum levels were detected by ELISA and dimethylarginine dimethylaminohydrolase (DDAH) -1/-2 mRNA-expression in peripheral mononuclear cells (PBMC) was analyzed by real time PCR. RESULTS: At baseline, the mean (±SD) arteriolar to venular diameter ratio (AVR) was impaired in obese (OA: 0.81±0.05) compared to lean subjects (AA: 0.87±0.07; EA: 0.94±0.05). The individual fitness levels correlated with AVR (rho=+0.66; P<0.001) and the training program improved AVR in all groups (P<0.001), normalising AVR in the obese (OA: 0.86±0.1). A training-induced arteriolar dilatation was found in OA (P=0.01), which was accompanied by a significant decrease of ADMA levels (0.56±0.12-0.46±0.12 µmoll(-1); P<0.028). DDAH-1 mRNA levels in PBMC increased in all groups (P<0.01). CONCLUSIONS: Cardiovascular fitness and body composition affect retinal vessel diameters. Regular exercise reverses the subclinical impairment of the retinal microvasculature in obesity by inducing retinal arteriolar dilatation. The NO/ADMA pathway may play a key role in the training-induced improvement of microvascular function, which has the potential to counteract progression of small vessel disease.

Enhancement of paclitaxel-mediated cytotoxicity in lung cancer cells by 17-allylamino geldanamycin: in vitro and in vivo analysis.

BACKGROUND: It has previously been demonstrated that 17-allylamino geldanamycin (17-AAG) enhances paclitaxel-mediated cytotoxicity and downregulates vascular endothelial factor expression in non-small cell lung cancer. This project was designed to evaluate the tumoricidal and antiangiogeneic effects of 17-AAG and paclitaxel in H358 non-small cell lung cancer cells grown as xenografts in nude mice. METHODS: In vitro cytotoxic drug combination effects were evaluated by (4, 5-dimethylthiazo-2-yl)-2, 5-diphenyl tetrazolium bromide-based proliferation assays. The combinations of 17-AAG and paclitaxel were administered intraperitoneally in nude mice bearing H358 tumor xenografts. Tumor volumes were measured weekly. Tumor expression of erbB2, vascular endothelial cell growth factor, von Willebrand factor (tumor microvasculature), and activated caspase 3 (apoptosis) were determined by immunohistochemistry. RESULTS: Five- to 22-fold enhancement of paclitaxel cytotoxicity was achieved by paclitaxel + 17-AAG combination that was paralleled with marked induction of apoptosis. This combination treatment profoundly suppressed tumor growth and significantly prolonged survival of mice bearing H358 xenografts. Immunohistochemical staining of tumor tissues indicated profound reduction of vascular endothelial cell growth factor expression associated with reduction of microvasculature in tumors treated with 17-AAG. Apoptotic cells were more abundant in tumors treated with 17-AAG + paclitaxel than in those treated with 17-AAG or paclitaxel alone. CONCLUSIONS: Concurrent exposure of H358 cells to 17-AAG and paclitaxel resulted in supraadditive growth inhibition effects in vitro and in vivo. Analysis of molecular markers of tumor tissues indicated that therapeutic drug levels could be achieved with this chemotherapy regimen leading to significant biological responses. Moreover, 17-AAG-mediated suppression of vascular endothelial cell growth factor production by tumor cells may contribute to the antitumor effects of this drug combination in vivo.

Effect of retroviral endostatin gene transfer on subcutaneous and intraperitoneal growth of murine tumors.

BACKGROUND: Inhibiting tumor angiogenesis is a promising new strategy for treating cancer. Difficulties with the stability, manufacture, and long-term administration of recombinant antiangiogenic proteins have prompted investigators to use gene therapy to generate these proteins in vivo. We investigated whether transfer of the gene encoding the angiogenesis inhibitor endostatin into the murine liver cell line NMuLi could inhibit tumor growth in vivo. METHODS: NMuLi cells were transduced with retroviral vectors containing the murine endostatin gene. The presence and function of endostatin in transduced cell supernatants were confirmed by competitive enzyme immunoassay and endothelial cell proliferation assays. Nude mice were given a subcutaneous or intraperitoneal injection with NMuLi cells, control transduced cells (NEF-null), or endostatin-transduced clones (NEF-Endo1 to 4) and were monitored for tumor growth. All statistical tests were two-sided. RESULTS: Supernatants from the clone secreting the lowest amount of endostatin (NEF-Endo4, 28 ng/mL) inhibited endothelial cell proliferation by 6% (95% confidence interval [CI] = 0% to 12%), and those from the clone secreting the highest amount (NEF-Endo1, 223 ng/mL) inhibited endothelial cell proliferation by 20% (95% CI = 13% to 27%). Increased levels of endostatin were detected in tumor lysates, but not serum, of mice given a subcutaneous injection of NEF-Endo1 cells. After 63 days, mice given a subcutaneous injection of parental NMuLi or NEF-null cells had tumor volumes of 2400 mm(3) (95% CI = 1478 mm(3) to 3300 mm(3)) and 2700 mm(3) (95% CI = 2241 mm(3) to 3144 mm(3)), respectively, compared with mean tumor volumes of less than 30 mm(3) in mice given an injection of NEF-Endo clones, a statistically significant difference (P<.001). After 123 days, all 16 mice given an intraperitoneal injection of parental NMuLi or NEF-null cells had died, compared with only three (9%) of 32 mice given an injection of NEF-Endo clones. CONCLUSIONS: Retroviral endostatin gene transfer leads to secretion of functional endostatin that is sufficiently active to inhibit tumor growth. Further studies of retroviral endostatin gene transfer for the treatment of cancer are warranted.

A mouse model of multiple endocrine neoplasia, type 1, develops multiple endocrine tumors.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant cancer syndrome, characterized primarily by multiple tumors in the parathyroid glands, endocrine pancreas, and anterior pituitary. Other tumors, including gastrinoma, carcinoid, adrenal cortical tumors, angiofibroma, collagenoma, and lipoma, also occur in some patients. Individuals with MEN1 almost always have loss-of-function mutations in the MEN1 gene on chromosome 11, and endocrine tumors arising in these patients usually show somatic loss of the remaining wild-type allele. To examine the role of MEN1 in tumor formation, a mouse model was generated through homologous recombination of the mouse homolog Men1. Homozygous mice die in utero at embryonic days 11.5-12.5, whereas heterozygous mice develop features remarkably similar to those of the human disorder. As early as 9 months, pancreatic islets show a range of lesions from hyperplasia to insulin-producing islet cell tumors, and parathyroid adenomas are also frequently observed. Larger, more numerous tumors involving pancreatic islets, parathyroids, thyroid, adrenal cortex, and pituitary are seen by 16 months. All of the tumors tested to date show loss of the wild-type Men1 allele, further supporting its role as a tumor suppressor gene.

Transcriptional activation of the proopiomelanocortin gene by cyclic AMP-responsive element binding protein.

The proopiomelanocortin (POMC) gene expressed in corticotrophs of the anterior pituitary encodes several biologically active peptides and is primarily under the positive control of hypophysiotropic factors (e.g. corticotropin releasing hormone). Using AtT20 cells as a model, we show that these factors increase levels of POMC primary RNA transcripts representative of a transcriptional activation of the gene. This effect is mimicked by several activators of the cAMP signaling pathway. Inhibition of protein synthesis with cycloheximide did not modify the CRH-induced increase in POMC hnRNA suggesting that these early effects are mediated by preexisting transcription factors. Using a reporter gene containing 706 bp of the POMC promoter region, we observe transcriptional activation with the same compounds, their effects being abolished when protein kinase A (PKA) is inactivated by a dominant inhibitory mutant. Promoter deletion analyses mapped an essential cAMP inducible element within the first exon of the POMC gene. This element (PTRE: TGACTAA) located at nucleotides +41/+47 was shown to bind the cAMP responsive element binding protein (CREB) by gel shift analyses and confers strong transcriptional activation by an expression vector coding a CREB-VP16 activator domain fusion protein. Further, expression of a dominant inhibitory mutant of CREB reduced cAMP stimulated transcription of the full length POMC promoter and the PTRE. Taken together, these results show that the major hypophysiotropic factors stimulate POMC transcription through a signaling cascade that involves PKA and CREB.

Discrete structural domains and cell-specific expression determine functional selectivity of the dopamine and norepinephrine transporters.

The successful generation and functional expression of a series of recombinant chimeric transporters, in which distinct functional properties of NET and DAT are exchanged, have allowed the assignment of a number of important functional properties of MPP+ and antidepressant-sensitive catecholamine transporters to specific domains within their primary structure. These studies are the first comprehensive structure-function analysis of members of the rapidly growing superfamily of Na+/Cl- carriers using chimeric transporters. This represents the first step in identifying the specific structural or regulatory determinants that differentiate NET and DAT. An appreciation of the potentially distinct sites for substrate recognition, translocation, and transport inhibition of NET and DAT may facilitate the development of more selective drugs for the treatment of stimulant addiction, human depression, and other affective disorders.

Ovarian steroid regulation of estrogen and progesterone receptor messenger ribonucleic acid in the anteroventral periventricular nucleus of the rat.

The anteroventral periventricular nucleus of the preoptic region (AVPV) represents a key site for hormonal feedback on gonadotropin secretion. It plays a critical role in the neural control of luteinizing hormone secretion and contains high densities of neurons that express receptors for estrogen and progesterone. In this study in situ hybridization was used to examine the expression of mRNAs encoding the estrogen (ER) and progesterone (PR) receptors in the AVPV during the estrous cycle. ER gene expression fluctuated during the cycle with the lowest levels of ER mRNA observed in animals killed on the afternoon of proestrus, and the highest levels present in animals killed during metestrus. This apparent inverse relationship between circulating levels of estradiol (E2) and ER mRNA levels in AVPV neurons was supported by the observation that treatment of ovariectomized rats with E2 suppressed expression of ER mRNA in the AVPV. The influence of progesterone (P4) on ER expression was less pronounced, but a significant increase in ER mRNA in the AVPV was detected 3 h after treatment with P4. In contrast, PR mRNA levels were highest in the AVPV during diestrus and lowest on the morning of proestrus suggesting that PR expression in the AVPV is regulated in a complex manner that may reflect the combined regulatory effects of E2 and P4. E2 treatment caused a dramatic induction of PR mRNA in the AVPV, but P4 did not affect PR mRNA expression acutely, although PR mRNA appears to be attenuated in the AVPV 27 h after P4 treatment. These findings suggest that ovarian steroid hormones regulate ER and PR gene expression in the AVPV during the estrous cycle, which may represent molecular events that contribute to cyclic changes in the responsiveness of AVPV neurons to steroid hormones.

Corticotropin-releasing hormone stimulates proopiomelanocortin transcription by cFos-dependent and -independent pathways: characterization of an AP1 site in exon 1.

The POMC gene, encoding a hormonal precursor protein, is primarily expressed in the pituitary in a tissue-specific manner. The POMC gene is transcriptionally regulated by a variety of hormones and neuropeptides and the second messengers cAMP and Ca++. Using the corticotrope-derived AtT20 cell line, we have previously shown that overexpression of cFos stimulates POMC transcription. The aim of this work was to analyze whether cFos directly interacts with the POMC gene in basal and corticotropin-releasing hormone (CRH) stimulated cells. Using progressively deleted POMC promoter sequences or heterologous promoter constructs coupled to the chloramphenicol acetyl transferase reporter gene, we demonstrate the existence of a major cFos- responsive sequence within the first exon of the POMC gene. This sequence, TGACTAA, appears functionally indistinguishable from the canonical AP1 binding site. When fused to a minimal promoter, this sequence confers inducibility by cFos and CRH. Gel shift analyses with CRH-stimulated AtT20 nuclear extracts or in vitro synthesized proteins revealed that this sequence efficiently binds Fos and Jun. Expression of c-fos anti-sense mRNA reduced CRH-stimulated POMC transcription, thus indicating that, at least in part, cFos mediates the effect of CRH on POMC transcription. However, deletion of this major exonic AP1 site from the POMC constructs greatly reduced the effect of c-fos overexpression but did not suppress POMC stimulation by CRH, indicating that CRH stimulates POMC transcription by one or more cFos-independent mechanism(s).

Cell-type-specific expression of catecholamine transporters in the rat brain.

The dopamine transporter (DAT) and norepinephrine transporter (NET) terminate catecholaminergic neurotransmission at synapses by high-affinity sodium-dependent reuptake into presynaptic terminals, and are the initial sites of action for drugs of abuse and antidepressants. In the present study, we used in situ hybridization combined with immunohistochemistry to study the distribution of DAT and NET mRNA in the adult rat brain. Cells were first immunolabeled with antisera directed against one of the catecholamine-synthetic enzymes, tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), or phenylethanolamine-N-methyltransferase (PNMT), in order to identify dopaminergic, noradrenergic, or epinephrine-containing cells. The immunolabeled cells were subsequently assayed for their ability to express catecholamine transporter mRNAs by in situ hybridization using either a rat DAT or NET cRNA probe. All dopaminergic cell groups of the mesencephalon contained high levels of DAT mRNA but only the A12 and A13 dopaminergic cell groups of the diencephalon appear to express detectable levels of DAT. All norepinephrine-containing cell bodies in the brainstem (locus coeruleus and lateral tegmentum) appear to express NET mRNA. In contrast, epinephrine-containing cell bodies of the brainstem do not appear to express NET mRNA, which raises the possibility that epinephrine may utilize a transporter that is distinct from the other bioactive amines, or may act as an endocrine regulator that does not require rapid reuptake mechanisms. Moreover, the cell-type-specific expression of catecholamine transporters suggests that DAT and NET gene expression may be closely linked to cellular mechanisms that specify transmitter phenotype. The termination of neurotransmission is a critical component of neural signaling and depends on the rapid removal of neurotransmitters from the synaptic cleft. Pharmacological evidence indicates that the action of monoamines at the synapse is terminated predominantly by rapid reuptake into presynaptic nerve endings via neurotransmitter-specific, high-affinity, Na(+)-dependent membrane transporter proteins. The cDNAs encoding distinct transporter proteins for the monoamines dopamine, norepinephrine, and serotonin have been cloned, expressed, and characterized in a variety of heterologous systems (Blakely et al., 1991; Giros et al., 1991; Hoffman et al., 1991; Kilty et al., 1991; Pacholczyk et al., 1991; Shimada et al., 1991; Usdin et al., 1991). Although the monoamine transporters share a high degree of sequence homology, they are distinguished by their monoamine substrate specificities and by their differential sensitivities to a wide spectrum of transport antagonists. For example, pharmacological agents that potently inhibit norepinephrine and serotonin transport, such as desmethylimipramine and citalopram, have little effect on the activity of the dopamine transporter (Javitch et al., 1983).(ABSTRACT TRUNCATED AT 400 WORDS)

Cloning and expression of a cocaine-sensitive rat dopamine transporter.

The action of dopamine and other monoamine neurotransmitters at synapses is terminated predominantly by high-affinity reuptake into presynaptic terminals by specific sodium-dependent neurotransmitter transport proteins. A complementary DNA encoding a rat dopamine transporter has been isolated that exhibits high sequence similarity with the previously cloned norepinephrine and gamma-aminobutyric acid transporters. Transient expression of the complementary DNA in HeLa cells confirms the cocaine sensitivity of this transporter.

Brain transections differentially alter lordosis and ear wiggling of 6-day-old rats.

Six-day-old male and female rats display lordosis and ear wiggling in response to tactile stimulation of the flanks and rump, without priming by exogenous estrogen. The involvement of various brain regions in these behaviors, which resemble components of adult female sexual behavior, was examined by making acute transections along the neuraxis from the olfactory tract to the medulla in 6-day-old rats. Four to 5 hr after the transection procedure, pups were tested for lordosis and ear wiggling. Lordosis was severely reduced or eliminated in pups with cuts through the hindbrain or diencephalon (above the level of the mammillary bodies) but was relatively unaffected by cuts through the posterior hypothalamus and rostral tegmentum and by cuts rostral to the anterior hypothalamus. Ear wiggling was disrupted by transections throughout the hindbrain and was facilitated only in females by transections throughout the forebrain (anterior to the mammillary bodies). These data suggest that facilitation from the hypothalamus is required for lordosis in the infant rat and the forebrain inhibitory systems for ear wiggling are functional in female infants by 6 days of age. Similarities and differences between the neural control of lordosis and ear wiggling in infant and adult rats suggest that the infant sex-like behaviors may be precursors of adult female sexual behavior.