Diabetes and Vascular Disease: Basic Concepts of Nitric Oxide Physiology, Endothelial Dysfunction, Oxidative Stress and Therapeutic Possibilities

The vascular manifestations associated with diabetes mellitus (DM) result from the dysfunction of several vascular physiology components mainly involving the endothelium, vascular smooth muscle and platelets. It is also known that hyperglycemia-induced oxidative stress plays a role in the development of this dysfunction. This review considers the basic physiology of the endothelium, especially related to the synthesis and function of nitric oxide. We also discuss the pathophysiology of vascular disease associated with DM. This includes the role of hyperglycemia in the induction of oxidative stress and the role of advanced glycation end-products. We also consider therapeutic strategies.

Bupivacaine 0.25% and methylene blue spread with epidural anesthesia in dog

Objective To evaluate the extent sensory and motor blocks produced by the epidural injection of different volumes of 0.25% bupivacaine (Bu) with methylene blue (MB), in dogs. Study design Prospective experimental trial. Animals Twenty healthy adult mongrel dogs, weighing 9.9 +/- 1.9 kg. Methods Dogs were randomly allocated into one of four groups that received 0.2, 0.4, 0.6 or 0.8 mL kg-1 of an epidural solution containing 0.25% Bu and MB. Sensory block was evaluated against time by pinching the tail, hind limb interdigital web, toenail bases and the skin over the vertebral dermatomes. Motor block was assessed by ataxia, hind limb weight-bearing ability and by loss of muscle tone of the tail and pelvic limbs. Data were collected at 2, 5, 10, 15 and 30 minutes after the end of epidural injection. After the final time point, dogs were euthanatized and laminectomies were conducted to expose the extent of the dural dye staining. Results The volumes 0.2, 0.4, 0.6 and 0.8 mL kg-1 of 0.25% Bu and MB blocked a mean of 5, 14.2, 20.2 and 21 dermatomes, respectively. The extent of the senory block increased up to a volume of 0.6 mL kg-1. Motor block was longer-lasting and more intense than sensory block. Complete dyeing of the spinal cord with MB was achieved in some dogs at 0.4 mL kg-1 and all dogs at 0.6 mL kg-1. Conclusions The volume of anesthetic injected into the epidural space plays an important role in the quality of the epidural anesthesia. At 0.25%, bupivacaine provided an efficient sensory block at 0.6 mL kg-1. Clinical relevance Relatively high volumes (0.6 mL kg-1) of 0.25%, BU and MB were needed to produce an effective sensory and motor block caudal to the umbilicus, but all spinal cord segments were reached by MB at this dose.

Morphological and Molecular Pathology of CCl(4)-Induced Hepatic Fibrosis in Connexin43-Deficient Mice

Gap junction channels, formed by connexins (Cx), are involved in the maintenance of tissue homeostasis, cell growth, differentiation, and development. Several studies have shown that Cx43 is involved in the control of wound healing in dermal tissue. However, it remains unknown whether Cx43 plays a role in the control of liver fibrogenesis. Our study investigated the roles of Cx43 heterologous deletion on carbon tetrachloride (CCl(4))-induced hepatic fibrosis in mice. We administered CCl(4) to both Cx43-deficient (Cx43(+/-)) and wild-type mice and examined hepatocellular injury and collagen deposition by histological and ultrastructural analyses. Serum biochemical analysis was performed to quantify liver injury. Hepatocyte proliferation was analyzed immunohistochemically. Protein and messenger RNA (mRNA) expression of liver connexins were evaluated using immunohistochemistry as well as immunoblotting analysis and quantitative real-time PCR. We demonstrated that Cx43(+/-) mice developed excessive liver fibrosis compared with wild-type mice after CCl(4)-induced chronic hepatic injury, with thick and irregular collagen fibers. Histopathological evaluation showed that Cx43(+/-) mice present less necroinflammatory lesions in liver parenchyma and consequent reduction of serum aminotransferase activity. Hepatocyte cell proliferation was reduced in Cx43(+/-) mice. There was no difference in Cx32 and Cx26 protein or mRNA expression in fibrotic mice. Protein expression of Cx43 increased in CCl(4)-treated mice, although with aberrant protein location on cytoplasm of perisinusoidal cells. Our results demonstrate that Cx43 plays an important role in the control and regulation of hepatic fibrogenesis. Microsc. Res. Tech. 74:421-429, 2011. (C) 2010 Wiley-Liss, Inc.

Expression of Homeobox Genes in Oral Squamous Cell Carcinoma Cell Lines Treated With All-Trans Retinoic Acid

Oral squamous cell carcinoma (OSCC) may arise from potentially malignant oral lesions. All-trans retinoic acid (atRA), which plays a role in cell growth and differentiation, has been studied as a possible chemotherapeutic agent in the prevention of this progression. While the mechanism by which atRA suppresses cell growth has not been completely elucidated, it is known that homeobox genes are atRA targets. To determine if these genes are involved in the atRA-mediated OSCC growth inhibition, PCR array was performed to evaluate the expression of 84 homeobox genes in atRA-sensitive SCC-25 cells compared to atRA-resistant SCC-9 cells following 7 days with atRA treatment. Results showed that the expression of 8 homeobox genes was downregulated and expression of 4 was upregulated in SCC-25 cells but not in SCC-9 cells. Gene expression levels were confirmed for seven of these genes by RT-qPCR. Expression of three genes that showed threefold downregulation was evaluated in SCC-25 cells treated with atRA for 3, 5, and 7 days. Three different patterns of atRA-dependent gene expression were observed. ALX1 showed downregulation only on day 7. DLX3 showed reduced expression on day 3 and further reduced on clay 7. TLX1 showed downregulation only on days 5 and 7. Clearly the expression of homeobox genes is modulated by atRA in OSCC cell lines. However, the time course of this modulation suggests that these genes are not direct targets of atRA mediating OSCC growth suppression. Instead they appear to act as downstream effectors of atRA signaling. J. Cell. Biochem. 111: 1437-1444, 2010. (C) 2010 Wiley-Liss, Inc.

Gender inequality, poverty and human development in Kenya: Main indicators, trends and limitations

Indicators of gender inequality, poverty and human development in Kenya are examined. Significant and rising incidence of absolute poverty occurs in Kenya and women are more likely to be in poverty than men. Female/male ratios in Kenyan decision-making institutions are highly skewed against women and they experience unfavourable enrolment ratios in primary, secondary and tertiary institutions. The share of income earned by women is much lower than men's share. General Kenyan indicators highlight declining GDP per capita, increased poverty rates especially for women, reduced life expectancy, a narrowing of the difference in female/male life expectancy rates, increased child mortality rates and an increase in the female child mortality rates. This deterioration results in an increased socio-economic burden on women, not adequately captured in the HPI, HDI, GDI and GEM. This paper advocates the use of household level gender disaggregated data because much gender inequality occurs in and emanates from the household level where culture plays a very important role in allocation of resources and decision-making. Because most human development indicators are aggregates or averages, they can be misleading. They need to be supplemented by distributional and disaggregated data as demonstrated in the Kenyan case. The importance is emphasised of studying coping mechanisms of household/families for dealing with economic hardship and other misfortunes, such AIDS.

Expression of transforming growth factor-beta 1, -beta 2, and -beta 3 in human developing teeth: Immunolocalization according to the odontogenesis phases

Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor that has several biological effects in vivo including control of cell growth and differentiation, cell migration, lineage determination, motility, adhesion, apoptosis, and synthesis and degradation of extracellular matrix, and TGF-beta plays an important role in regulating tissue repair and regeneration. Our study analyzed the participation of TGF-beta 1, -beta 2, and -beta 3 in the different stages of morphogenesis and differentiation of human developing dental organ using immunobistochemistry. The maxillae and mandibles of 10 human embryos ranging from 8 to 23 weeks of gestation were employed, according to the approval of the ethical committee. Our study revealed that the TGF-beta subunits-beta 1, beta 2, and beta 3 were present in the various stages of tooth development, but the expression varied according to the differentiation stage, tissue, and TGF-beta subunit. Our results indicated that TGF-beta 1 is closely related to differentiation of enamel organ and initiation of matrix secretion, TGF-beta 2 to cellular differentiation, and TGF-beta 3 to mineral maturation matrix.

CCR5 Down-regulates Osteoclast Function in Orthodontic Tooth Movement

During orthodontic tooth movement, there is local production of chemokines and an influx of leukocytes into the periodontium. CCL5 plays an important role in osteoclast recruitment and activation. This study aimed to investigate whether the CCR5-receptor influences these events and, consequently, orthodontic tooth movement. An orthodontic appliance was placed in wild-type mice (WT) and CCR5-deficient mice (CCR5(-/-)). The expression of mediators involved in bone remodeling was evaluated in periodontal tissues by Real-time PCR. The number of TRAP-positive osteoclasts and the expression of cathepsin K, RANKL, and MMP13 were significantly higher in CCR5(-/-). Meanwhile, the expression of two osteoblastic differentiation markers, RUNX2 and osteocalcin, and that of bone resorption regulators, IL-10 and OPG, were lower in CCR5(-/-). Analysis of the data also showed that CCR5(-/-) exhibited a greater amount of tooth movement after 7 days of mechanical loading. The results suggested that CCR5 might be a down-regulator of alveolar bone resorption during orthodontic movement.

Substance P and NK-1R expression in oral precancerous epithelium

The objectives of this study were to investigate the presence and distribution of substance P and neurokinin 1 receptor in oral premalignant epithelium and their relation with the presence of dysplasia, and to analyze whether the expression of substance P can be considered an early oncogenic event in oral carcinogenesis. Substance P and neurokinin I receptor expression was immunohistochemically studied in 83 oral carcinomas and adjacent nontumor epithelia. The presence and degree of epithelial dysplasia was assessed according to WHO criteria. The nuclear, cytoplasmic, and membrane expression of substance P and the cytoplasmic and membrane expression of neurokinin 1 receptor were assessed in tumor and adjacent non-tumor epithelium. Nuclear and cytoplasmic expression of substance P in non-tumor epithelium was significantly associated with the presence of epithelial dysplasia (p<0.001) and carcinoma in. situ (p=0.021). Nuclear, cytoplasmic, and membrane expressions of substance P in non-tumor epithelium were significantly (p<0.001) associated with its expression in the corresponding tumor. These findings suggest that substance P plays a role in early oral carcinogenesis by promoting the proliferation and growth of premalignant fields.

Role of the spinal cord heme oxygenase-carbon monoxide-cGMP pathway in the nociceptive response of rats

The aim of the present study was to investigate the role of the spinal cord heme oxygenase (HO)-carbon monoxide (CO)-soluble guanylate cyclase (sGC)-cGMP pathway in nociceptive response of rats to the formalin experimental nociceptive model. Animals were handled and adapted to the experimental environment for a few days before the formalin test was applied. For the formalin test 50 mu l of a 1% formalin solution was injected subcutaneously in the dorsal surface of the right hind paw. Following injections, animals were observed for I h and flinching behavior was measured as the nociceptive response. Thirty min before the test, rats were pretreated with intrathecal injections with the HO inhibitor, zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG) or heme-lysinate, which is known to induce the HO pathway. Control animals were treated with vehicles. We observed a significant increase in nociceptive response of rats treated with ZnDPBG, and a drastic reduction of flinching nociceptive behavioral response in the heme-lysinate treated animals. Furthermore, the HO pathway seems to act via cGMP, since methylene blue (a sGC inhibitor) prevented the reduction of flinching nociceptive behavioral response caused by heme-lysinate. These findings strongly indicate that the HO pathway plays a spinal antinociceptive role during the formalin test, acting via cGMP. (C) 2007 Elsevier B.V. All rights reserved.

Involvement of the heme oxygenase-carbon monoxide-cGMP pathway in the nociception induced by acute painful stimulus in rats

Heme oxygenase-carbon monoxide-cGMP (HO-CO-cGMP) pathway has been reported to be involved in peripheral and spinal modulation of inflammatory pain. However, the involvement of this pathway in the modulation of acute painful stimulus in the absence of inflammation remains unknown. Thus, we evaluated the involvement of the HO-CO-cGMP pathway in nociception by means the of analgesia index (AI) in the tail flick test. Rats underwent surgery for implantation of unilateral guide cannula directed toward the lateral ventricle and after the recovery period (5-7 days) were subjected to the measures of baseline tail flick test Animals were divided into groups to assess the effect of intracerebroventricular administration (i.c.v.) of the following compounds: ZnDPBG (HO inhibitor) or vehicle (Na(2)CO(3)), heme-lysinate (substrate overload) or vehicle (L-lysine), or the selective inhibitor of soluble guanilate cyclase ODQ or vehicle (DMSO 1%) following the administration of heme-lysinate or vehicle. Heme overload increased AI, indicating an antinociceptive role of the pathway. This response was attenuated by i.c.v. pretreatment with the HO inhibitor ZnDPBG. In addition, this effect was dependent on cGMP activity, since the pretreatment with ODQ blocked the increase in the AI. Because CO produces most of its actions via cGMP, these data strongly imply that CO is the HO product involved in the antinociceptive response. This modulation seems to be phasic rather than tonic, since i.c.v. treatment with ZnDPBG or ODQ did not alter the AI. Therefore, we provide evidence consistent with the notion that HO-CO-cGMP pathway plays a key phasic antinociceptive role modulating noninflammatory acute pain. (C) 2011 Elsevier B.V. All rights reserved.

Ionotropic glutamatergic receptors in the rostral medullary raphe modulate hypoxia and hypercapnia-induced hyperpnea

It has been suggested that the medullary raphe (MR) plays a key role in the physiological responses to hypoxia and hypercapnia. We assessed the role of ionotropic glutamate receptors in the rostral MR (rMR) in the respiratory responses to hypoxia and hypercapnia by measuring pulmonary ventilation (V(E)) and body temperature (Tb) of male Wistar rats before and after microinjecting Kynurenic acid (KY, an ionotropic glutamate receptors antagonist, 0.1 mM) into the rMR followed by 60 min of hypoxia (7% O(2)) or hypercapnia exposure (7% CO(2)). Compared to the control group, the ventilatory response to hypoxia was attenuated in animals treated with KY intra-rMR, however the ventilatory response to hypercapnia increased significantly. No differences in Tb among groups were observed during hypoxia or hypercapnia. These data suggest that the glutamate acting on ionotropic receptors in the rMR exerts an excitatory modulation on hyperventilation induced by hypoxia but an inhibitory modulation on the hypercapnia-induced hyperpnea. (C) 2010 Elsevier B.V. All rights reserved.

Kisspeptin Regulates Prolactin Release through Hypothalamic Dopaminergic Neurons

Prolactin (PRL) is tonically inhibited by dopamine (DA) released from neurons in the arcuate and periventricular nuclei. Kisspeptin plays a pivotal role in LH regulation. In rodents, kisspeptin neurons are found mostly in the anteroventral periventricular and arcuate nuclei, but the physiology of arcuate kisspeptin neurons is not completely understood. We investigated the role of kisspeptin in the control of hypothalamic DA and pituitary PRL secretion in adult rats. Intracerebroventricular kisspeptin-10 (Kp-10) elicited PRL release in a dose-dependent manner in estradiol (E2)-treated ovariectomized rats (OVX+E2), whereas no effect was found in oil-treated ovariectomized rats (OVX). Kp-10 increased PRL release in males and proestrous but not diestrous females. Associated with the increase in PRL release, intracerebroventricular Kp-10 reduced Fos-related antigen expression in tyrosine hydroxylase-immunoreactive (ir) neurons of arcuate and periventricular nuclei in OVX+E2 rats, with no effect in OVX rats. Kp-10 also decreased 3,4-dihydroxyphenylacetic acid concentration and 3,4-dihydroxyphenylacetic acid-DA ratio in the median eminence but not striatum in OVX+E2 rats. Double-label immunofluorescence combined with confocal microscopy revealed kisspeptin-ir fibers in close apposition to and in contact with tyrosine hydroxylase-ir perikarya in the arcuate. In addition, Kp-10 was not found to alter PRL release from anterior pituitary cell cultures regardless of E2 treatment. We provide herein evidence that kisspeptin regulates PRL release through inhibition of hypothalamic dopaminergic neurons, and that this mechanism is E2 dependent in females. These findings suggest a new role for central kisspeptin with possible implications for reproductive physiology. (Endocrinology 151: 3247-3257, 2010)

Propyretic role of the locus coeruleus nitric oxide pathway

Nitric oxide has been reported to modulate fever in the brain. However, the sites where NO exerts this modulation remain somewhat unclear. Locus coeruleus (LC) neurons express not only nitric oxide synthase (NOS) but also soluble guanylyl cyclase (sGC). In the present study, we evaluated in vivo and ex vivo the putative role of the LC NO-cGMP pathway in fever. To this end, deep body temperature was measured before and after pharmacological modulations of the pathway. Moreover, nitrite/nitrate (NOx) and cGMP levels in the LC were assessed. Conscious rats were microinjected within the LC with a non-selective NOS inhibitor (NG-monomethyl-l-arginine acetate), a NO donor (NOC12), a sGC inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) or a cGMP analogue (8-bromo-cGMP) and injected intraperitoneally with endotoxin. Inhibition of NOS or sGC before endotoxin injection significantly increased the latency to the onset of fever. During the course of fever, inhibition of NOS or sGC attenuated the febrile response, whereas microinjection of NOC12 or 8-bromo-cGMP increased the response. These findings indicate that the LC NO-cGMP pathway plays a propyretic role. Furthermore, we observed a significant increase in NOx and cGMP levels, indicating that the febrile response to endotoxin is accompanied by stimulation of the NO-cGMP pathway in the LC.

Evidence for the involvement of matrix metalloproteinases in the cardiovascular effects produced by nicotine

Nicotine plays a role in smoking-associated cardiovascular diseases, and may upregulate matrix metalloproteinase (MMP)-2 and MMP-9. We examined whether nicotine induces the release of MMP-2 and MMP-9 by rat smooth muscle cells (SMC), and whether doxycycline (non-selective MMP inhibitor) inhibits the vascular effects produced by nicotine. SMC were incubated with nicotine 0, 50, and 150 nM for 48 h. MMP-2 and MMP-9 levels in the cell supernatants were determined by gelatin zymography. The acute changes in mean arterial pressure caused by nicotine 2 mu mol/kg (or saline) were assessed in rats pretreated with doxycycline (or saline). We also examined whether doxcycline (30 mg/Kg, i.p., daily) modifies the effects of nicotine (10 mg/kg/day; 4 weeks) on the endothelium-dependent relaxations of rat aortic rings. Aortic MMP-2 levels were assessed by gelatin zymography. Aortic gelatinolytic activity was assessed using a gelatinolytic activity kit. MMP-2 and MMP-9 levels increased in the supernatant of SMC cells incubated with nicotine 150 nM (P<0.05) but not with 50 nM. Nicotine (2 mu mol/kg) produced lower increases in the mean arterial pressure in rats pretreated with doxycycline than those found in rats pretreated with saline (26 +/- 4 vs. 37 +/- 4 mmHg, respectively; P<0.05). Nicotine impaired of the endothelium-dependent responses to acetylcholine, and treatment with doxycycline increased the potency (pD2) by approximately 25% (P<0.05). While we found no significant differences in aortic MMP-2 levels, nicotine significantly increased gelatinolytic activity (P<0.05). These findings suggest that nicotine produces cardiovascular effects involving MMPs. It is possible that MMPs inhibition may counteract the effects produced by nicotine. (C) 2009 Elsevier B.V. All rights reserved.

Serotonergic mechanisms on breathing modulation in the rat locus coeruleus

The locus coeruleus (LC) is a noradrenergic nucleus that plays an important role in the ventilatory response to hypercapnia. This nucleus is densely innervated by serotonergic fibers and contains high density of serotonin (5-HT) receptors, including 5-HT(1A) and 5-HT(2). We assessed the possible modulation of respiratory response to hypercapnia by 5-HT, through 5-HT(1A) and 5-HT(2) receptors, in the LC. To this end, we determined the concentrations of 5-HT and its metabolite 5-hydroxyindole-3-acetic acid (5-HIAA) in the LC after hypercapnic exposure. Pulmonary ventilation (V(E), plethysmograph) was measured before and after unilateral microinjection (100 nL) of WAY-100635 (5-HT(1A) antagonist, 5.6 and 56 mM), 8-OHDPAT (5-HT(1A/7) agonist, 7 and 15 mM), Ketanserin (5-HT(2A) antagonist, 3.7 and 37 mM), or (+/-)-2,5-dimethoxy-4-iodoamphetaminehydrochloride (DOI; 5-HT(2A) agonist, 6.7 and 67 mM) into the LC, followed by a 60-min period of 7% CO(2) exposure. Hypercapnia increased 5-HTIAA levels and 5-HIAA/5-HT ratio within the LC. WAY-100635 and 8-OHDPAT intra-LC decreased the hypercapnic ventilatory response due to a lower tidal volume. Ketanserin increased CO(2) drive to breathing and DOI caused the opposite response, both acting on tidal volume. The current results provide evidence of increased 5-HT release during hypercapnia in the LC and that 5-HT presents an inhibitory modulation of the stimulatory role of LC on hypercapnic ventilatory response, acting through postsynaptic 5-HT(2A) receptors in this nucleus. In addition, hypercapnic responses seem to be also regulated by presynaptic 5-HT(1A) receptors in the LC.