Andreas Vesalius as a renaissance innovative neuroanatomist: his 5th centenary of birth / Andreas Vesalius como neuroanatomista inovador renascentista: seu 5° centenário de nascimento

Andreas Vesalius (1514-1564) is considered the Father of Modern Anatomy, and an authentic representative of the Renaissance. His studies, founded on dissection of human bodies, differed from Galeno, who based his work on dissection of animals, constituted a notable scientific advance. Putting together science and art, Vesalius associated himself to artists of the Renaissance, and valued the images of the human body in his superb work De Humani Corporis Fabrica.This paper aims to honor this extraordinary European Renaissance physician and anatomist, who used aesthetic appeal to bind text and illustration, science and art. His achievements are highlighted, with an especial attention on neuroanatomy. Aspects about his personal life and career are also focused.

Andreas Vesalius (1514-1564) é considerado o Pai da Anatomia Moderna e um autêntico representante da Renascença. Seus estudos, baseados na dissecação de corpos humanos, diferiam dos de Galeno, que baseava seu trabalho em dissecação de animais, constituiu-se em um notável avanço científico. Reunindo ciência e arte, Vesalius associou-se a artistas da Renascença e valorizou as imagens do corpo humano em seu soberbo trabalho De Humani Corporis Fabrica. Este artigo visa honrar esse extraordinário médico e anatomista da Renascença europeia, que fez uso do apelo estético para coligar texto e ilustração, ciência e arte. Suas realizações são realçadas, com atenção especial na neuroanatomia. Também são colocados em foco aspectos da sua vida pessoal e de sua carreira.

Genetic characterization of Toxoplasma gondii isolates from eared doves (Zenaida auriculata) in Brazil / Caracterização genética de isolados de Toxoplasma gondii de pombos (Zenaida auriculata) no Brasil

Eared doves (Zenaida auriculata), which are common in urban, rural and wild areas in many regions of Brazil, are frequently prey for domestic cats. Therefore Toxoplasma gondii isolates obtained from doves may reflect greater environmental diversity than those from other hosts. The aim of the present study was to evaluate T. gondii seroprevalence, isolate and genotype strains from Z. auriculata. Serum and tissue samples were collected from 206 doves for use in the modified agglutination test (MAT) and mouse bioassay. The prevalence of T. gondii antibodies in the doves was 22.3% (46/206), with titers ranging from 16 to 4096, and T. gondii strains were isolated from 12 of these doves. Five genotypes were detected by means of PCR-RFLP, including ToxoDB genotypes #1, #6, #17 and #65, and one genotype that had not previously been described (ToxoDB#182). This was the first report on isolation of T. gondii from Z. auriculata. This study confirmed the genetic diversity of T. gondii isolates and the existence of clonal type II (ToxoDB genotype #1) in Brazil.

Pombos silvestres (Zenaida auriculata), comuns em áreas urbanas, rurais e selvagens em muitas regiões do Brasil, são frequentemente predados por gatos domésticos. Sendo assim, os isolados de T. gondii obtidos de pombos podem refletir uma maior diversidade ambiental do que os outros hospedeiros. O objetivo do presente estudo foi avaliar a soroprevalência, isolar e genotipar T. gondii de Z. auriculata. Amostras de soro e tecido foram coletadas de 206 pombos para o teste de aglutinação modificado (MAT) e o bioensaio em camundongos. A prevalência de anticorpos contra T. gondii em pombos foi 22,3% (46/206), com títulos variando de 16 a 4096, e T. gondii foi isolado de 12 pombos. Cinco genótipos foram detectados por PCR-RFLP, incluindo os genótipos ToxoDB #1, #6, #17, #65 e um genótipo não descrito anteriormente (ToxoDB#182). Esse é o primeiro relato de isolamento de T. gondii de Z. auriculata. Este estudo também confirmou a diversidade dos isolados de T. gondii e a presença de tipo clonal II (ToxoDB #1) no Brasil.

Arginine induces GH gene expression by activating NOS/NO signaling in rat isolated hemi-pituitaries

The amino acid arginine (Arg) is a recognized secretagogue of growth hormone (GH), and has been shown to induce GH gene expression. Arg is the natural precursor of nitric oxide (NO), which is known to mediate many of the effects of Arg, such as GH secretion. Arg was also shown to increase calcium influx in pituitary cells, which might contribute to its effects on GH secretion. Although the mechanisms involved in the effects of Arg on GH secretion are well established, little is known about them regarding the control of GH gene expression. We investigated whether the NO pathway and/or calcium are involved in the effects of Arg on GH gene expression in rat isolated pituitaries. To this end, pituitaries from approximately 170 male Wistar rats (~250 g) were removed, divided into two halves, pooled (three hemi-pituitaries) and incubated or not with Arg, as well as with different pharmacological agents. Arg (71 mM), the NO donor sodium nitroprusside (SNP, 1 and 0.1 mM) and a cyclic guanosine monophosphate (cGMP) analogue (8-Br-cGMP, 1 mM) increased GH mRNA expression 60 min later. The NO acceptor hemoglobin (0.3 µM) blunted the effect of SNP, and the combined treatment with Arg and L-NAME (a NO synthase (NOS) inhibitor, 55 mM) abolished the stimulatory effect of Arg on GH gene expression. The calcium channel inhibitor nifedipine (3 µM) also abolished Arg-induced GH gene expression. The present study shows that Arg directly induces GH gene expression in hemi-pituitaries isolated from rats, excluding interference from somatostatinergic neurons, which are supposed to be inhibited by Arg. Moreover, the data demonstrate that the NOS/NO signaling pathway and calcium mediate the Arg effects on GH gene expression.

Endothelial nitric oxide synthase gene polymorphisms [G894T] in diabetes mellitus in Egypt

Diabetic nephropathy [DN] is one of the major microvascular complications of diabetes. Genetic predisposition has been implicated in DN. The eNOS protein synthesizes nitric oxide constitutively via a reaction including the conversion of L-arginine to L-citrulline, which involves the transfer of five electrons provided by nicotinamide adenine dinucleotide phosphate The aim of this study is to evaluate the association of G894T polymorphisms of endothelial nitric oxide synthase [eNOS] gene with the development of diabetic nephropathy [DN] among Egyptian patients with type 1,2 diabetes mellitus in Egypt. Study subjects comprised 86 patients of type 2 diabetes with nephropathy,23 patients of type 1 diabetes with nephropathy and 46 patients of type 2 diabetes without nephropathy. G894T genotypes was determined by SSP- PCR analysis. Mutant T allele, GT and TT genotypes of G894TSNP had no significant frequencies in type 1.2 diabetic patients with nephropathy compared to those without nephropathy. These findings indicate that G894T polymorphism of eNOS gene may be not considered as genetic risk factors for DN among Egyptian type 1, 2 diabetic patients. T2DM: type 2 diabetes mellitus-DN: diabetic nephropathy eNOS: Endothelial nitric oxide synthase:- SNP: single nucleotide polymorphism- SSP-PCR: sequence specific primer- polymerase chain reaction

Nitric oxide-mediated pathogenesis during nicotine and alcohol consumption.

Nitric oxide (NO) is formed by different cell types in response to a variety of physiological and patho-physiological stimuli. The intake of nicotine and/or alcohol has patho-physiological effects on organ function, and the progression of alcohol-/tobacco-related diseases seem to be directly influenced by NO-mediated mechanisms. Nicotine has an adverse influence on blood vessel functionality, repair and maintenance. Chronic nicotine exposure augments atherosclerosis by enhancing the production of proinflammatory cytokines by macrophages which then activate atherogenic NF-kB target genes in aortic lesions. Alcohol produces NO which speeds up the apoptosis of neutrophils. Alcohol sensitizes the liver to endotoxemic shock. Nitrosative stress and increased basal levels of NO contribute to tumour growth. The progression of disease seems to be directed via a definite NO-mediated mechanism. This review gives an insight into how intake of tobacco and alcohol may affect quality of life.

Association of the 894G>T polymorphism of the endothelial constitutive nitric oxide synthase gene with unstable angina

The 894G>T polymorphism of the endothelial constitutive nitric oxide synthase gene consists of the substitution of a guanine base by a thymine at the 894th nucleotide of the gene. An association of this polymorphism with acute coronary syndromes has been described, only when in combination with other polymorphisms of this gene. The aim of the present study was to search for an association between this polymorphism and unstable angina in a southern Brazilian population. In a case-control study, 156 patients (group 1 (N = 83): unstable angina, group 2 (N = 73): stable angina) were genotyped by PCR and digestion of the product. Univariate analysis demonstrated that the minimal luminal diameter and the degree of stenosis of the culprit lesion differed between groups (P = 0.006 and 0.005, respectively). In addition, the frequencies of the T allele and of the T allele carriers (combined TT and TG genotypes) were significantly higher in the group with unstable angina (41.6 vs 28.8 percent; P = 0.025, Pearson chi-square test, and 73.5 vs 45.2 percent; P = 0.001, Pearson chi-square test, respectively). Multivariate logistic regression showed that the frequency of the T allele carriers was the only variable with a predictive value for unstable angina, when controlled for the other variables (6.1 (95 percent CI = 2.55-14.43); P < 0.001). Thus, in a homogenous group of patients, the endothelial constitutive nitric oxide synthase 894G>T polymorphism was associated with unstable angina. We suggest that this polymorphism may be a genetic risk factor for unstable angina.

Interleukin-10 endogenously expressed in microglia prevents lipopolysaccharide-induced neurodegeneration in the rat cerebral cortex in vivo

A degree of brain inflammation is required for repair of damaged tissue, but excessive inflammation causes neuronal cell death. Here, we observe that IL-10 is expressed in LPS-injected rat cerebral cortex, contributing to neuronal survival. Cells immunopositive for IL-10 were detected as early as 8 h post-injection and persisted for up to 3 d, in parallel with the expression of IL-1beta, TNF-alpha, and iNOS. Double immunofluorescence staining showed that IL-10 expression was localized mainly in activated microglia. Next, we examined the neuroprotective effects of IL-10 using IL-10 neutralizing antibody (IL-10NA). Blockade of IL-10 action caused a significant loss of neurons both 3 d and 7 d after LPS injection. Further, the induction of mRNA species encoding IL-1beta, TNF-alpha, and iNOS, reactive oxygen species (ROS) production, and NADPH oxidase activation, increased after co-injection of LPS and IL-10NA, compared to the levels seen after injection of LPS alone. Taken together, these results clearly suggest that LPS-induced endogenous expression of IL-10 in microglia contributes to neuronal survival by inhibiting brain inflammation.

Effect of berberine on the mRNA expression of nitric oxide synthase (NOS) in rat corpus cavernosum / 华中科技大学学报（医学）（英德文版）

In order to further investigate the mechanisms of action of berberine (Ber), we assessed the effects of Ber on the mRNA expression of nitric oxide synthases (NOS) in rat corpus cavernosum. After incubation with Ber for 1 or 3 h respectively, the levels of NOS mRNA were examined by reverse transcription polymerase chain reaction (RT-PCR). Our results showed that there were iNOS and eNOS mRNA expressions in rat corpus cavernosum. Ber enhanced eNOS mRNA expression in rat penis, but exhibited no effect on the expression of iNOS mRNA (P > 0.05). The present study indicated that the relaxation of Ber involved the NO-cGMP signal transduction pathway. The enhancing effect of Ber on eNOS mRNA expression might associated with its relaxation of corpus cavernosum.

Fasting-induced Down-regulation of NADPH-diaphorase in the Magnocellular PVN of Rats

In this study, we examined if glucocorticoids are required for the fasting-induced decrease of neuronal nitric oxide synthase (nNOS) in the magnocellular division of the paraventricular nucleus (PVN). Rats were adrenalectomized, subjected to 48 h of food deprivation with/without dexamethasone (5 mg/ kg, 4 subcutaneous injections with 12 h intervals), and the brain slices were processed for NADPH-diaphorase (NADPH- d) staining, a histochemical marker for nNOS in neuronal cells. In food deprived adrenalectomized rats, but not in free fed intact rats, dexamethasone significantly decreased NADPH-d staining in the magnocellular PVN. We previously reported that food deprivation decreases nNOS in the magnocellular PVN of intact rats. Thus, the present results together with our previous report suggest that although glucocorticoids are required for fasting-induced nNOS down-regulation in the magnocellular PVN, glucocorticoids may not be directly involved and some other molecular signals produced by food deprivation may play a pivotal role over glucocorticoid in the regulatory pathway for nNOS expression in this brain region.

Protein kinase A mediates microglial activation induced by plasminogen and gangliosides

In the injured brain, microglia is known to be activated and produce proinflammatory mediators such as interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS). We investigated the role of protein kinase A (PKA) in microglial activation by both plasminogen and gangliosides in rat primary microglia and in the BV2 immortalized murine microglial cell line. Both plasminogen and gangliosides induced IL-1beta, TNF-alpha and iNOS mRNA expression, and that this expression was inhibited by the addition of the PKA inhibitors, KT5720 and H89. Both plasminogen and gangliosides activated PKA and increased the DNA binding activity of the cAMP response element- binding protein (CREB). Furthermore, KT5720 and H89 reduced the DNA binding activities of CREB and NF-kappaB in plasminogen-treated cells. These results suggest that PKA plays an important role in plasminogen and gangliosides- induced microglial activation.

Progresión de la poliquistosis renal autosómica dominante. Influencia de polimorfismos de genes de sintasa endotelial del óxido nítrico (ecNOS) y del sistema renina-angiotensina / Progression of autosomic dominant polycystic kidney disease. Influence of endothelial NO synthase (ecNOS) and renin angiotensin system gene polymorphisms

Glomerular filtration rate decline (GFRd) is variable in autosomic dominant polycystic kidney disease (ADPKD). In 88 ADPKD patients, GFRd was assessed by 1/S(Cr) and compared with the association to AT1A1166C (AT1R), AGTM235T (angiotensinogen) and ecNOSGlu298Asp (NO endothelial synthase) polymorphisms. Age at S(Cr) values of 2 and 6 mg/dl were assumed as beginning of progressive phase (A2) and end-stage-renal disease (A6), respectively. Polymorphisms were studied by PCR-RFLP. The group as a whole showed GFRd (ml/min/year) of 6.9+/-0.5; A2 and A6 of 48.9+/-1.3 and 55.0+/-1.4 years and mean arterial pressure of 111.2+/-1.2 mmHg. When A6 was considered, two populations were defined (< or = and > 55 years). In < or = 55 (assumed as PKD1 phenotype) (n=42), A2 and A6 of the AT1 1166CC genotype were 36.0+/-1.2 and 41.4+/-0.9 years vs AA-AC (42.8+/-1.0 and 47.5+/-0.8, p<0.001). A2 and A6 of the ecNOS298Asp/Asp genotype were 34.8+/-1.5 and 41.1+/-0.6 years vs. Glu/Glu-Glu/Asp (42.4+/-0.9 and 47.1+/-0.8, p<0.02). In AGT235TT genotype, GFRd was 12.4+/-2.2 ml/min/year vs MM-MT (7.9+/-0.7, p<0.03). This difference was also observed when all ADPKD patients were considered (TT: 11.02+/-1.5 vs. MM-MT: 6.44+/-0.5 ml/ min/year, p<0.003). AT1 1166CC and ecNOS 298Asp/Asp are associated with earlier A2 and A6 whereas AGT 235TT induce twofold increase in GFRd, suggesting that RAS and ecNOS are involved in ADPKD progression.

La velocidad de progresión (VdP) de la poliquistosis renal autosómica dominante (PQRAD) es variable.Estudiamos la asociación de los polimorfismos AGTM235T (angiotensinógeno), AT1A1166C(ATR1) y ecNOSGlu298Asp (NO sintasa endotelial) con la VdP en 88 pacientes. VdP fue estimada por 1/Crplvs edad. Consideramos edades de Crpl 2 y 6 mg/dl como comienzo de progresión (E2) y arribo a insuficienciarenal crónica terminal (E6), respectivamente. Los polimorfismos se estudiaron por PCR-RFLP. El grupo en sutotalidad presentó VdP (ml/min/año) de 6.9±0.5, E2 y E6 de 48.9±1.3 y 55.0±1.4 años y tensión arterial media(TAM) de 111.2±1.2 mmHg. Según E6 observamos dos grupos (≤ y > a 55 años). En ≤ 55 (fenotipo PKD1,n=42), E2 y E6 del genotipo CC de AT1A1166C fueron 36.0±1.2 y 41.4±0.9 años vs. AA-AC (42.8±1.0 y 47.5±0.8, p < 0.001). E2 y E6 del genotipo ecNOS298Asp/Asp fueron 34.8±1.5 y 41.1±0.6 años vs. Glu/Glu-Glu/Asp (42.4±0.9 y 47.1±0.8, p < 0.02). En el genotipo AGT235TT, la VdP fue 12.4±2.2 ml/min/año vs. MM-MT (7.9±0.7, p < 0.03). Esta diferencia también se observó cuando analizamos todos los pacientes PQRAD (TT: 11.02±1.5 vs. MM-MT: 6.44±0.5 ml/min/año, p < 0.003). Los genotipos AT1 1166CC y ecNOS 298Asp/Asp anticipan E2 y E6 mientras que AGT235TT duplica VdP, sugiriendo la participación del sistema renina angiotensina y NO sintasaendotelial en la progresión de la PQRAD.

Soluble factor from tumor cells induces heme oxygenase-1 by a nitric oxide-independent mechanism in murine peritoneal macrophages

Tumor target-derived soluble secretary factor has been known to influence macrophage activation to induce nitric oxide (NO) production. Since heme oxigenase-1 (HO-1) is induced by a variety of conditions associated with oxidative stress, we questioned whether soluble factor from tumor cells induces HO-1 through NO-dependent mechanism in macrophages. We designated this factor as a tumor-derived macrophage-activating factor (TMAF), because of its ability to activate macrophages to induce iNOS. Although TMAF alone showed modest activity, TMAF in combination with IFN-gamma significantly induced iNOS expression and NO synthesis. Simultaneously, TMAF induced HO-1 and this induction was slightly augmented by IFN-gamma. Surprisingly, however, induction of HO-1 by TMAF was not inhibited by the treatment with the highly selective iNOS inhibitor, 1400 W, indicating that TMAF induces the HO-1 enzyme by a NO-independent mechanism. While rIFN-gamma alone induced iNOS, it had no effect on HO-1 induction by itself. Collectively, the current study reveals that soluble factor from tumor target cells induces HO-1 enzyme in macrophages. However, overall biological significance of this phenomenon remains to be determined.

Genes and diabetic retinopathy.

Several recent studies have provided evidence that good diabetes control is important to prevent diabetic retinopathy. However, some groups of patients develop diabetic retinopathy despite good control and others escape retinopathy despite poor control. This suggests the role of genetic factors in susceptibility to retinopathy. This article reviews the role of genetic factors in determining diabetic retinopathy.

Expression and Regulation of Endothelial Nitric Oxide Synthase by Vascular Endothelial Growth Factor in ECV 304 Cells

Nitric oxide (NO) seems to play a pivotal role in the vascular endothelial growth factor (VEGF)-induced endothelial cell proliferation. This study was designed to investigate the role and intracellular signal pathway of endothelial nitric oxide synthase (eNOS) activation induced by VEGF. ECV 304 cells were treated with betaVEGF(165) and then cell proliferation, eNOS protein and mRNA expression levels were analyzed to elucidate the functional role of eNOS in cell proliferation induced by VEGF. After exposure of cells to betaVEGF(165) , eNOS activity and cell growth were increased by approximately two-fold in the betaVEGF(165) -treated cells compared to the untreated cells. In addition, VEGF stimulated eNOS expression at both the mRNA and protein levels in a dose-dependent manner. Phosphatidylinositol-3 kinase (PI-3K) inhibitors were used to assess PI-3K involvement in eNOS regulation. LY294002 was found to attenuate VEGF-stimulated eNOS expression. Wortmannin was not as effective as LY294002, but the reduction effect was detectable. Cells activated by VEGF showed increased ERK1/2 levels. Moreover, the VEGF-induced eNOS expression was reduced by the PD98059, MAPK pathway inhibitor. This suggests that eNOS expression might be regulated by PI-3K and the ERK1/2 signaling pathway. In conclusion, betaVEGF(165) induces ECV 304 cell proliferation via the NO produced by eNOS. In addition, eNOS may be regulated by the PI-3K or mitogen-activated protein kinase pathway.

Decreased nitric oxide synthesis in rats with chronic renal failure

The present study was aimed at investigating whether an altered role of nitric oxide (NO) is involved in chronic renal failure (CRF). Rats were subjected to 5/6 nephrectomy and kept for 6 weeks to induce CRF. On the experimental day, after measurement of arterial pressure under anesthesia, the arterial blood was collected, and thoracic aorta and kidney were rapidly taken. NO metabolites (NOx) were determined in the plasma, urine, aorta and kidney. The expression of NO synthase (NOS) isozymes was determined in the kidney and aorta by Western blot analysis. The expression of NOS mRNA in the glomeruli was also determined by RT-PCR. There were significant increases in arterial pressure and serum creatinine levels in CRF. Urine NOx levels were decreased in CRF, whereas plasma NOx levels were not altered. Aorta and kidney tissue NOx levels were also decreased in CRF. The expression of endothelial constitutive (ec) and inducible (i) isoforms of NOS proteins was decreased in the kidney and aorta in CRF. Accordingly, the expression of ecNOS and iNOS mRNA was decreased in the glomeruli in CRF. In conclusion, NO synthesis is decreased in the kidney and vasculature of CRF rats.

Decreased nitric oxide synthesis in rats with chronic renal failure

The present study was aimed at investigating whether an altered role of nitric oxide (NO) is involved in chronic renal failure (CRF). Rats were subjected to 5/6 nephrectomy and kept for 6 weeks to induce CRF. On the experimental day, after measurement of arterial pressure under anesthesia, the arterial blood was collected, and thoracic aorta and kidney were rapidly taken. NO metabolites (NOx) were determined in the plasma, urine, aorta and kidney. The expression of NO synthase (NOS) isozymes was determined in the kidney and aorta by Western blot analysis. The expression of NOS mRNA in the glomeruli was also determined by RT-PCR. There were significant increases in arterial pressure and serum creatinine levels in CRF. Urine NOx levels were decreased in CRF, whereas plasma NOx levels were not altered. Aorta and kidney tissue NOx levels were also decreased in CRF. The expression of endothelial constitutive (ec) and inducible (i) isoforms of NOS proteins was decreased in the kidney and aorta in CRF. Accordingly, the expression of ecNOS and iNOS mRNA was decreased in the glomeruli in CRF. In conclusion, NO synthesis is decreased in the kidney and vasculature of CRF rats.

Regulaçäo da expressão da óxido nítrico sintase induzível (iNOS) pelo hormônio de crescimento (GH): Estudo in vitro e em modelo animal de glumerulosclerose (camundongo trangênico para GH) / Regulation of the inducible nitric oxide synthases (INOS) expression by growth hormone (GH): In vitro study and in transgenic mouse model of glomerulosclerosis

Estudamos a regulação de enzimas da via de metabolização da L-arginina (óxido nítrico sintase induzível, iNOS, ornitina descarboxilase, ODC, e ornitina aminotransferase, OAT) pelo hormônio de crescimento (GH) em células mesangiais e em córtex renal de camundongos bGH. Estes animais apresentam concentração sérica elevada de GH e desenvolvem glomerulosclerose (GS) progressiva. Há várias evidências da participação do GH no desenvolvimento de GS. Demonstrou-se também uma associação entre o acúmulo de matrix extracelular (MEC) nos glomérulos e a expressão aumentada de iNOS, OAT e ODC. Nós demonstramos, pela primeira vez, uma ligação entre o GH e a iNOS em células mesangiais, além de uma expressão aumentada desta enzima em córtex renal de camundongo bGH com GS severa...

Neuronal and endothelial nitric oxide synthase gene knockout mice

Targeted disruption of the neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) genes has led to knockout mice that lack these isoforms. These animal models have been useful to study the roles of nitric oxide (NO) in physiologic processes. nNOS knockout mice have enlarged stomachs and defects in the inhibitory junction potential involved in gastrointestinal motility. eNOS knockout mice are hypertensive and lack endothelium-derived relaxing factor activity. When these animals are subjected to models of focal ischemia, the nNOS mutant mice develop smaller infarcts, consistent with a role for nNOS in neurotoxicity following cerebral ischemia. In contrast, eNOS mutant mice develop larger infarcts, and show a more pronounced hemodynamic effect of vascular occlusion. The knockout mice also show that nNOS and eNOS isoforms differentially modulate the release of neurotransmitters in various regions of the brain. eNOS knockout mice respond to vessel injury with greater neointimal proliferation, confirming that reduced NO levels seen in endothelial dysfunction change the vessel response to injury. Furthermore, eNOS mutant mice still show a protective effect of female gender, indicating that the mechanism of this protection cannot be limited to upregulation of eNOS expression. The eNOS mutant mice also prove that eNOS modulates the cardiac contractile response to ß-adrenergic agonists and baseline diastolic relaxation. Atrial natriuretic peptide, upregulated in the hearts of eNOS mutant mice, normalizes cGMP levels and restores normal diastolic relaxation.