Nitric Oxide Synthase Knockout Mice Research Articles

Endothelin Receptor A Antagonism and Fetal Growth in Endothelial Nitric Oxide Synthase Gene Knockout Maternal and Fetal Mice.

Fetal growth restriction (FGR) is commonly associated with perinatal morbidity and mortality. Nitric oxide (NO) deficiency increases endothelin-1 (ET-1) production, and this increased ET-1 may contribute to the pathophysiology of NO deficiency-induced FGR. Using an endothelial NO synthase knockout mouse model of FGR, we sought to determine (a) the relative importance of maternal versus conceptus (fetal and placental) NO deficiency and (b) the contribution of ET-1 to the pathogenesis of FGR in this model. Fetal growth restriction occurred both with NO-deficient conceptuses in the setting of maternal NO production and with maternal NO deficiency in the setting of NO-producing conceptuses. Placental ET-1 expression was increased in NO-deficient dams, ET receptor A (ETA) production increased in endothelial nitric oxide synthase(+/-) placentas, and antagonism of ETA prevented FGR. These results demonstrate that both maternal and conceptus NO deficiency can contribute to FGR and suggest a role for ETA antagonists as therapeutic agents in FGR.

Endothelial AMPK activation induces mitochondrial biogenesis and stress adaptation via eNOS-dependent mTORC1 signaling

Metabolic stress sensors like AMP-activated protein kinase (AMPK) are known to confer stress adaptation and promote longevity in lower organisms. This study demonstrates that activating the metabolic stress sensor AMP-activated protein kinase (AMPK) in endothelial cells helps maintain normal cellular function by promoting mitochondrial biogenesis and stress adaptation. To better define the mechanisms whereby AMPK promotes endothelial stress resistance, we used 5-aminoimidazole-4-carboxamide riboside (AICAR) to chronically activate AMPK and observed stimulation of mitochondrial biogenesis in wild type mouse endothelium, but not in endothelium from endothelial nitric oxide synthase knockout (eNOS-null) mice. Interestingly, AICAR-enhanced mitochondrial biogenesis was blocked by pretreatment with the mammalian target of rapamycin complex 1 (mTORC1) inhibitor, rapamycin. Further, AICAR stimulated mTORC1 as determined by phosphorylation of its known downstream effectors in wild type, but not eNOS-null, endothelial cells. Together these data indicate that eNOS is needed to couple AMPK activation to mTORC1 and thus promote mitochondrial biogenesis and stress adaptation in the endothelium. These data suggest a novel mechanism for mTORC1 activation that is significant for investigations in vascular dysfunction.

Prostaglandin I2 Receptor Agonism Preserves β-Cell Function and Attenuates Albuminuria Through Nephrin-Dependent Mechanisms

Discovery of common pathways that mediate both pancreatic β-cell function and end-organ function offers the opportunity to develop therapies that modulate glucose homeostasis and separately slow the development of diabetes complications. Here, we investigated the in vitro and in vivo effects of pharmacological agonism of the prostaglandin I2 (IP) receptor in pancreatic β-cells and in glomerular podocytes. The IP receptor agonist MRE-269 increased intracellular 3',5'-cyclic adenosine monophosphate (cAMP), augmented glucose-stimulated insulin secretion (GSIS), and increased viability in MIN6 β-cells. Its prodrug form, selexipag, augmented GSIS and preserved islet β-cell mass in diabetic mice. Determining that this preservation of β-cell function is mediated through cAMP/protein kinase A (PKA)/nephrin-dependent pathways, we found that PKA inhibition, nephrin knockdown, or targeted mutation of phosphorylated nephrin tyrosine residues 1176 and 1193 abrogated the actions of MRE-269 in MIN6 cells. Because nephrin is important to glomerular permselectivity, we next set out to determine whether IP receptor agonism similarly affects nephrin phosphorylation in podocytes. Expression of the IP receptor in podocytes was confirmed in cultured cells by immunoblotting and quantitative real-time PCR and in mouse kidneys by immunogold electron microscopy, and its agonism 1) increased cAMP, 2) activated PKA, 3) phosphorylated nephrin, and 4) attenuated albumin transcytosis. Finally, treatment of diabetic endothelial nitric oxide synthase knockout mice with selexipag augmented renal nephrin phosphorylation and attenuated albuminuria development independently of glucose change. Collectively, these observations describe a pharmacological strategy that posttranslationally modifies nephrin and the effects of this strategy in the pancreas and in the kidney.

Neurovascular Protective Function of Endothelial Nitric Oxide - Recent Advances.

In the central nervous system endothelial nitric oxide (NO) is an essential molecule responsible for the preservation of the functional integrity of the neurovascular unit. NO causes vasodilatation and is an important inhibitor of platelet aggregation, smooth muscle cell proliferation, and white blood cell adhesion. In addition, endothelium-derived NO exerts anti-inflammatory and pro-angiogenic effects. More recently, it has been recognized that endothelial NO modulates the expression and processing of amyloid precursor protein in cerebrovascular endothelium and neuronal tissue. Studies in endothelial NO synthase (eNOS) knockout mice indicate that endothelial NO functions as a neurovascular protective molecule during aging. Indeed, genetic inactivation of eNOS exacerbates the detrimental effects of aging on cerebrovascular, microglial, and neuronal functions as well as on cognition. These findings suggest that the preservation of healthy endothelium and normal function of eNOS might be important therapeutic targets. Because the beneficial effects of NO are mostly mediated by the activation of guanylate cyclase/cyclic GMP signaling, inhibitors of phosphodiesterase isoforms, or activation of this signaling with exercise, may offer therapeutic opportunities in the prevention and treatment of aging-induced cognitive decline and Alzheimer's disease. Most recent advances in understanding the molecular mechanisms linking loss of endothelial NO with cognitive decline will be discussed in this review. (Circ J 2016; 80: 1499-1503).

Sildenafil Therapy Normalizes the Aberrant Metabolomic Profile in the Comt(-/-) Mouse Model of Preeclampsia/Fetal Growth Restriction.

Preeclampsia (PE) and fetal growth restriction (FGR) are serious complications of pregnancy, associated with greatly increased risk of maternal and perinatal morbidity and mortality. These complications are difficult to diagnose and no curative treatments are available. We hypothesized that the metabolomic signature of two models of disease, catechol-O-methyl transferase (COMT−/−) and endothelial nitric oxide synthase (Nos3−/−) knockout mice, would be significantly different from control C57BL/6J mice. Further, we hypothesised that any differences in COMT−/− mice would be resolved following treatment with Sildenafil, a treatment which rescues fetal growth. Targeted, quantitative comparisons of serum metabolic profiles of pregnant Nos3−/−, COMT−/− and C57BL/6J mice were made using a kit from BIOCRATES. Significant differences in 4 metabolites were observed between Nos3−/− and C57BL/6J mice (p < 0.05) and in 18 metabolites between C57BL/6J and COMT−/− mice (p < 0.05). Following treatment with Sildenafil, only 5 of the 18 previously identified differences in metabolites (p < 0.05) remained in COMT−/− mice. Metabolomic profiling of mouse models is possible, producing signatures that are clearly different from control animals. A potential new treatment, Sildenafil, is able to normalize the aberrant metabolomic profile in COMT−/− mice; as this treatment moves into clinical trials, this information may assist in assessing possible mechanisms of action.

The impact of a human IGF-II analog ([Leu27]IGF-II) on fetal growth in a mouse model of fetal growth restriction.

Enhancing placental insulin-like growth factor (IGF) availability appears to be an attractive strategy for improving outcomes in fetal growth restriction (FGR). Our approach was the novel use of [Leu27]IGF-II, a human IGF-II analog that binds the IGF-II clearance receptor IGF-IIR in fetal growth-restricted (FGR) mice. We hypothesized that the impact of [Leu27]IGF-II infusion in C57BL/6J (wild-type) and endothelial nitric oxide synthase knockout (eNOS−/−; FGR) mice would be to enhance fetal growth and investigated this from mid- to late gestation; 1 mg·kg−1·day−1 [Leu27]IGF-II was delivered via a subcutaneous miniosmotic pump from E12.5 to E18.5. Fetal and placental weights recorded at E18.5 were used to generate frequency distribution curves; fetuses <5th centile were deemed growth restricted. Placentas were harvested for immunohistochemical analysis of the IGF system, and maternal serum was collected for measurement of exogenously administered IGF-II. In WT pregnancies, [Leu27]IGF-II treatment halved the number of FGR fetuses, reduced fetal(P = 0.028) and placental weight variations (P = 0.0032), and increased the numbers of pups close to the mean fetal weight (131 vs. 112 pups within 1 SD). Mixed-model analysis confirmed litter size to be negatively correlated with fetal and placental weight and showed that [Leu27]IGF-II preferentially improved fetal weight in the largest litters, as defined by number. Unidirectional 14CMeAIB transfer per gram placenta (System A amino acid transporter activity) was inversely correlated with fetal weight in [Leu27]IGF-II-treated WT animals (P < 0.01). In eNOS−/− mice, [Leu27]IGF-II reduced the number of FGR fetuses(1 vs. 5 in the untreated group). The observed reduction in FGR pup numbers in both C57 and eNOS−/− litters suggests the use of this analog as a means of standardizing and rescuing fetal growth, preferentially in the smallest offspring.

The Cardioprotective Effects of Late-Phase Remote Preconditioning of Trauma Depends on Neurogenic Pathways and the Activation of PKC and NF-κB (But Not iNOS) in Mice

A superficial abdominal surgical incision elicits cardioprotection against cardiac ischemia-reperfusion (I/R) injury in mice. This process, called remote preconditioning of trauma (RPCT), has both an early and a late phase. Previous investigations have demonstrated that early RPCT reduces cardiac infarct size by 80% to 85%. We evaluated the cardioprotective and molecular mechanisms of late-phase RPCT in a murine I/R injury model. Wild-type mice, bradykinin (BK) 2 receptor knockout mice, 3M transgenic mice (nuclear factor κB [NF-κb] repressor inhibitor of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha [IκBα((S32A, S36A, Y42F))]), and inducible nitric oxide synthase (iNOS) knockout mice were analyzed using a previously established I/R injury model. A noninvasive abdominal surgical incision was made 24 hours prior to I/R injury and the infarct size was determined at 24 hours post-I/R injury. The results indicated that a strong cardioprotective effect occurred during late-phase RPCT (58.42% ± 1.89% sham vs 29.41% ± 4.00% late RPCT, mean area of the infarct divided by the mean area of the risk region; P ≤ .05; n = 10). Furthermore, pharmacological intervention revealed the involvement of neurogenic signaling in the beneficial effects of late RPCT via sensory and sympathetic thoracic nerves. Pharmacological experiments in transgenic mice-implicated BK receptors, β-adrenergic receptors, protein kinase C, and NF-κB but not iNOS signaling in the cardioprotective effects of late RPCT. Late RPCT significantly decreased myocardial infarct size via neurogenic transmission and various other signaling pathways. This protective mechanism differentiates late and early RPCT. This study describes a new cardiac I/R injury prevention method and refines the concept of RPCT.

STAT1 signaling within macrophages is required for antifungal activity against Cryptococcus neoformans.

Cryptococcus neoformans, the predominant etiological agent of cryptococcosis, is an opportunistic fungal pathogen that primarily affects AIDS patients and patients undergoing immunosuppressive therapy. In immunocompromised individuals, C. neoformans can lead to life-threatening meningoencephalitis. Studies using a virulent strain of C. neoformans engineered to produce gamma interferon (IFN-γ), denoted H99γ, demonstrated that protection against pulmonary C. neoformans infection is associated with the generation of a T helper 1 (Th1)-type immune response and signal transducer and activator of transcription 1 (STAT1)-mediated classical (M1) macrophage activation. However, the critical mechanism by which M1 macrophages mediate their anti-C. neoformans activity remains unknown. The current studies demonstrate that infection with C. neoformans strain H99γ in mice with macrophage-specific STAT1 ablation resulted in severely increased inflammation of the pulmonary tissue, a dysregulated Th1/Th2-type immune response, increased fungal burden, deficient M1 macrophage activation, and loss of protection. STAT1-deficient macrophages produced significantly less nitric oxide (NO) than STAT1-sufficient macrophages, correlating with an inability to control intracellular cryptococcal proliferation, even in the presence of reactive oxygen species (ROS). Furthermore, macrophages from inducible nitric oxide synthase knockout mice, which had intact ROS production, were deficient in anticryptococcal activity. These data indicate that STAT1 activation within macrophages is required for M1 macrophage activation and anti-C. neoformans activity via the production of NO.

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect.

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these strategies is to examine their effects on heart function. There are several techniques to measure in vivo cardiac mechanics (e.g., echocardiography, pressure/volume relations, etc.). Compared to echocardiography, real-time left ventricular (LV) pressure/volume analysis via catheterization is more precise and insightful in assessing LV function. Additionally, LV pressure/volume analysis provides the ability to instantaneously record changes during manipulations of contractility (e.g., β-adrenergic stimulation) and pathological insults (e.g., ischemia/reperfusion injury). In addition to the maximum (+dP/dt) and minimum (-dP/dt) rate of pressure change in the LV, an accurate assessment of LV function via several load-independent indexes (e.g., end systolic pressure volume relationship and preload recruitable stroke work) can be attained. Heart rate has a significant effect on LV contractility such that an increase in the heart rate is the primary mechanism to increase cardiac output (i.e., Bowditch effect). Thus, when comparing hemodynamics between experimental groups, it is necessary to have similar heart rates. Furthermore, a hallmark of many cardiomyopathy models is a decrease in contractile reserve (i.e., decreased Bowditch effect). Consequently, vital information can be obtained by determining the effects of increasing heart rate on contractility. Our and others data has demonstrated that the neuronal nitric oxide synthase (NOS1) knockout mouse has decreased contractility. Here we describe the procedure of measuring LV pressure/volume with increasing heart rates using the NOS1 knockout mouse model.

Facilitation of corticostriatal transmission following pharmacological inhibition of striatal phosphodiesterase 10A: role of nitric oxide-soluble guanylyl cyclase-cGMP signaling pathways.

The striatum contains a rich variety of cyclic nucleotide phosphodiesterases (PDEs), which play a critical role in the regulation of cAMP and cGMP signaling. The dual-substrate enzyme PDE10A is the most highly expressed PDE in striatal medium-sized spiny neurons (MSNs) with low micromolar affinity for both cyclic nucleotides. Previously, we have shown that systemic and local administration of the selective PDE10A inhibitor TP-10 potently increased the responsiveness of MSNs to cortical stimulation. However, the signaling mechanisms underlying PDE10A inhibitor-induced changes in corticostriatal transmission are only partially understood. The current studies assessed the respective roles of cAMP and cGMP in the above effects using soluble guanylyl cyclase (sGC) or adenylate cyclase (AC) specific inhibitors. Cortically evoked spike activity was monitored in urethane-anesthetized rats using in vivo extracellular recordings performed proximal to a microdialysis probe during local infusion of vehicle, the selective sGC inhibitor ODQ, or the selective AC inhibitor SQ 22536. Systemic administration of TP-10 (3.2 mg/kg) robustly increased cortically evoked spike activity in a manner that was blocked following intrastriatal infusion of ODQ (50 μm). The effects of TP-10 on evoked activity were due to accumulation of cGMP, rather than cAMP, as the AC inhibitor SQ was without effect. Consistent with these observations, studies in neuronal NO synthase (nNOS) knock-out (KO) mice confirmed that PDE10A operates downstream of nNOS to limit cGMP production and excitatory corticostriatal transmission. Thus, stimulation of PDE10A acts to attenuate corticostriatal transmission in a manner largely dependent on effects directed at the NO-sGC-cGMP signaling cascade.

Lack of neuronal nitric oxide synthase results in attention deficit hyperactivity disorder-like behaviors in mice.

Nitric oxide (NO) is an important molecule for the proper development and function of the central nervous system. In this study, we investigated the behavioral alterations in the neuronal NO synthase knockout mice (NOS1 KO) with a deficient NO production mechanism in the brain, characterizing it as a potential rodent model for attention deficit hyperactivity disorder (ADHD). NOS1 KO exhibited higher locomotor activity than their wildtype counterparts in a novel environment, as measured by open field (OF) test. In a 2-way active avoidance paradigm (TWAA), we found sex-dependent effects, where male KO displayed deficits in avoidance and escape behavior, sustained higher incidences of shuttle crossings, and higher incidences of intertrial interval crossings, suggesting learning, and/or performance impairments. On the other hand, female KO demonstrated few deficits in TWAA. Molsidomine (MSD), a NO donor, rescued TWAA deficits in male KO when acutely administered before training. In a passive avoidance paradigm, KO of both sexes displayed significantly shorter step-through latencies after training. Further, abnormal spontaneous motor activity rhythms were found in the KO during the dark phase of the day, indicating dysregulation of rhythmic activities. These data indicate that NOS1 KO mimics certain ADHD-like behaviors and could potentially serve as a novel rodent model for ADHD.

Epidermal growth factor receptor signaling and the progression of diabetic nephropathy.

Epidermal growth factor receptor signaling and the progression of diabetic nephropathy.

Alterations of urinary metabolite profile in model diabetic nephropathy

Countering the diabetes pandemic and consequent complications, such as nephropathy, will require better understanding of disease mechanisms and development of new diagnostic methods. Animal models can be versatile tools in studies of diabetic renal disease when model pathology is relevant to human diabetic nephropathy (DN). Diabetic models using endothelial nitric oxide synthase (eNOS) knock-out mice develop major renal lesions characteristic of human disease. However, it is unknown whether they can also reproduce changes in urinary metabolites found in human DN. We employed Type 1 and Type 2 diabetic mouse models of DN, i.e. STZ-eNOS−/− C57BLKS and eNOS−/− C57BLKS db/db, with the goal of determining changes in urinary metabolite profile using proton nuclear magnetic resonance (NMR). Six urinary metabolites with significantly lower levels in diabetic compared to control mice have been identified. Specifically, major changes were found in metabolites from tricarboxylic acid (TCA) cycle and aromatic amino acid catabolism including 3-indoxyl sulfate, cis-aconitate, 2-oxoisocaproate, N-phenyl-acetylglycine, 4-hydroxyphenyl acetate, and hippurate. Levels of 4-hydroxyphenyl acetic acid and hippuric acid showed the strongest reverse correlation to albumin-to-creatinine ratio (ACR), which is an indicator of renal damage. Importantly, similar changes in urinary hydroxyphenyl acetate and hippurate were previously reported in human renal disease. We demonstrated that STZ-eNOS−/− C57BLKS and eNOS−/− C57BLKS db/db mouse models can recapitulate changes in urinary metabolome found in human DN and therefore can be useful new tools in metabolomic studies relevant to human pathology.

Endothelial dysfunction abrogates the efficacy of normobaric hyperoxia in stroke.

Hyperoxia has been uniformly efficacious in experimental focal cerebral ischemia. However, pilot clinical trials have showed mixed results slowing its translation in patient care. To explain the discordance between experimental and clinical outcomes, we tested the impact of endothelial dysfunction, exceedingly common in stroke patients but under-represented in experimental studies, on the neuroprotective efficacy of normobaric hyperoxia. We used hyperlipidemic apolipoprotein E knock-out and endothelial nitric oxide synthase knock-out mice as models of endothelial dysfunction, and examined the effects of normobaric hyperoxia on tissue perfusion and oxygenation using high-resolution combined laser speckle and multispectral reflectance imaging during distal middle cerebral artery occlusion. In normal wild-type mice, normobaric hyperoxia rapidly and significantly improved tissue perfusion and oxygenation, suppressed peri-infarct depolarizations, reduced infarct volumes, and improved neurological function. In contrast, normobaric hyperoxia worsened perfusion in ischemic brain and failed to reduce infarct volumes or improve neurological function in mice with endothelial dysfunction. These data suggest that the beneficial effects of hyperoxia on ischemic tissue oxygenation, perfusion, and outcome are critically dependent on endothelial nitric oxide synthase function. Therefore, vascular risk factors associated with endothelial dysfunction may predict normobaric hyperoxia nonresponders in ischemic stroke. These data may have implications for myocardial and systemic circulation as well.

Inhibition of kidney proximal tubular glucose reabsorption does not prevent against diabetic nephropathy in type 1 diabetic eNOS knockout mice.

Background and ObjectiveSodium glucose cotransporter 2 (SGLT2) is the main luminal glucose transporter in the kidney. SGLT2 inhibition results in glycosuria and improved glycaemic control. Drugs inhibiting this transporter have recently been approved for clinical use and have been suggested to have potential renoprotective benefits by limiting glycotoxicity in the proximal tubule. We aimed to determine the renoprotective benefits of empagliflozin, an SGLT2 inhibitor, independent of its glucose lowering effect.Research Design and MethodsWe induced diabetes using a low dose streptozotocin protocol in 7–8 week old endothelial nitric oxide (eNOS) synthase knockout mice. We measured fasting blood glucose on a monthly basis, terminal urinary albumin/creatinine ratio. Renal histology was assessed for inflammatory and fibrotic changes. Renal cortical mRNA transcription of inflammatory and profibrotic cytokines, glucose transporters and protein expression of SGLT2 and GLUT1 were determined. Outcomes were compared to diabetic animals receiving the angiotensin receptor blocker telmisartan (current best practice).ResultsDiabetic mice had high matched blood glucose levels. Empagliflozin did not attenuate diabetes-induced albuminuria, unlike telmisartan. Empagliflozin did not improve glomerulosclerosis, tubular atrophy, tubulointerstitial inflammation or fibrosis, while telmisartan attenuated these. Empagliflozin did not modify tubular toll-like receptor-2 expression in diabetic mice. Empagliflozin did not reduce the upregulation of macrophage chemoattractant protein-1 (MCP-1), transforming growth factor β1 and fibronectin mRNA observed in the diabetic animals, while telmisartan decreased transcription of MCP-1 and fibronectin. Empagliflozin increased GLUT1 mRNA expression and telmisartan increased SGLT2 mRNA expression in comparison to untreated diabetic mice. However no significant difference was found in protein expression of GLUT1 or SGLT2 among the different groups.ConclusionHence SGLT2 inhibition does not have renoprotective benefits independent of glucose lowering.

Exercise Training Induces Mitochondrial Biogenesis and Glucose Uptake in Subcutaneous Adipose Tissue Through eNOS-Dependent Mechanisms

Insulin resistance and obesity are associated with a reduction of mitochondrial content in various tissues of mammals. Moreover, a reduced nitric oxide (NO) bioavailability impairs several cellular functions, including mitochondrial biogenesis and insulin-stimulated glucose uptake, two important mechanisms of body adaptation in response to physical exercise. Although these mechanisms have been thoroughly investigated in skeletal muscle and heart, few studies have focused on the effects of exercise on mitochondria and glucose metabolism in adipose tissue. In this study, we compared the in vivo effects of chronic exercise in subcutaneous adipose tissue of wild-type (WT) and endothelial NO synthase (eNOS) knockout (eNOS(-/-)) mice after a swim training period. We then investigated the in vitro effects of NO on mouse 3T3-L1 and human subcutaneous adipose tissue-derived adipocytes after a chronic treatment with an NO donor: diethylenetriamine-NO (DETA-NO). We observed that swim training increases mitochondrial biogenesis, mitochondrial DNA content, and glucose uptake in subcutaneous adipose tissue of WT but not eNOS(-/-) mice. Furthermore, we observed that DETA-NO promotes mitochondrial biogenesis and elongation, glucose uptake, and GLUT4 translocation in cultured murine and human adipocytes. These results point to the crucial role of the eNOS-derived NO in the metabolic adaptation of subcutaneous adipose tissue to exercise training.

Applanation tonometry in mice: a novel noninvasive technique to assess pulse wave velocity and arterial stiffness.

Arterial stiffening is the root cause of a range of cardiovascular complications, including myocardial infarction, left ventricular hypertrophy, stroke, renal failure, dementia, and death, and a hallmark of the aging process. The most important in vivo parameter of arterial stiffness is pulse wave velocity (PWV). Clinically, PWV is determined noninvasively using applanation tonometry. Unlike the clinical value of arterial stiffness and PWV, techniques to determine PWV in mice are scarce. The only way to determine aortic PWV noninvasively in the mouse is by using ultrasound echo Doppler velocimetry. It is a fast, efficient, and accurate technique, but the required tools are expensive and technically complex. Here, we describe the development and validation of a novel technique to assess carotid-femoral PWV noninvasively in mice. This technique is based on applanation tonometry as used clinically. We were able to establish a reproducible reference value in wild-type mice (3.96±0.05 m/s) and to detect altered carotid-femoral PWV values in endothelial nitric oxide synthase knockout mice (4.66±0.05 m/s; P<0.001 compared with control), and in mice sedated with sodium pentobarbital (2.89±0.17 m/s; P<0.001 compared with control). Also, carotid-femoral PWV was pharmacologically modulated and measured in a longitudinal experiment with endothelial nitric oxide synthase knockout mice to demonstrate the applicability of this technique. In general, applanation tonometry can be used to measure carotid-femoral PWV noninvasively in mice. The experimental setup is simple, and the technical requirements are basic, making this technique readily implementable in any mouse model-based research facility interested in arterial stiffness.

The role of gaseous neurotransmitters in the antinociceptive effects of morphine during acute thermal pain

Treatment with a carbon monoxide-releasing molecule (tricarbonyldichlororuthenium(II) dimer, CORM-2) or a classical inducible heme oxygenase (HO-1) inducer (cobalt protoporphyrin IX, CoPP) enhanced the antinociceptive effects of morphine during chronic pain but the role played by these compounds in acute thermal nociception was not evaluated. The effects of CORM-2 and CoPP treatments on the local antinociceptive actions of morphine and their interactions with nitric oxide during acute pain were evaluated by using wild type (WT), neuronal (nNOS-KO) or inducible (iNOS-KO) nitric oxide synthase knockout mice and assessing their thermal nociception to a hot stimulus with the hot plate test. Our results showed that the absence of nNOS or iNOS genes did not alter licking and jumping responses nor the antinociceptive effects produced by morphine indicating that the local thermal inhibitory effects produced by this drug in the absence of inflammation or injury are not mediated by the nitric oxide pathway triggered by nNOS or iNOS enzymes. Moreover, while the systemic administration of CORM-2 or CoPP inhibited licking and jumping latencies in all genotypes, these treatments only enhanced the local inhibition of jumping latencies produced by morphine in WT and nNOS-KO mice which effects were reversed by the peripheral administration of an HO-1 inhibitor. These data indicate that the co-administration of morphine with CORM-2 or CoPP produced remarkable local antinociceptive effects in WT and nNOS-KO mice and reveal that a significant interaction between carbon monoxide and nitric oxide systems occurs on the local antinociceptive effects produced by morphine during acute thermal nociception.

Stem cell factor is a potent endothelial permeability factor.

Although stem cell factor (SCF) has been shown to play a critical role in hematopoiesis, gametogenesis, and melanogenesis, the function of SCF in the regulation of vascular integrity has not been studied. We demonstrated that SCF binds to and activates the cKit receptor in endothelial cells, thereby increasing the internalization of vascular endothelial-cadherin and enhancing extravasation of dyes to a similar extent as vascular endothelial growth factor. SCF-mediated cKit activation in endothelial cells enhanced the phosphorylation of endothelial nitric oxide (NO) synthase via the phosphoinositide 3-kinase/Akt signaling pathway and subsequently increased the production of NO. Inhibition of endothelial NO synthase expression and NO synthesis using small interfering RNA knockdown and chemical inhibitors substantially diminished the ability of SCF to increase the internalization of vascular endothelial-cadherin and in vitro endothelial permeability. SCF-induced increase in extravasation of the dyes was abrogated in endothelial NO synthase knockout mice, which indicates that endothelial NO synthase-mediated NO production was responsible for the SCF-induced vascular leakage. Furthermore, we demonstrated that the expression of SCF and cKit was significantly higher in the retina of streptozotocin-injected diabetic mice than in the nondiabetic control animals. Depletion of SCF by intravitreous injection of anti-SCF-neutralizing immunoglobulin G significantly prevented vascular hyperpermeability in the retinas of streptozotocin-injected diabetic mice. Our data reveal that SCF disrupts the endothelial adherens junction and enhances vascular leakage, as well as suggest that anti-SCF/cKit therapy may hold promise as a potential therapy for the treatment of hyperpermeable vascular diseases.

Podocyte-Specific VEGF-A Gain of Function Induces Nodular Glomerulosclerosis in eNOS Null Mice

VEGF-A and nitric oxide are essential for glomerular filtration barrier homeostasis and are dysregulated in diabetic nephropathy. Here, we examined the effect of excess podocyte VEGF-A on the renal phenotype of endothelial nitric oxide synthase (eNOS) knockout mice. Podocyte-specific VEGF(164) gain of function in eNOS(-/-) mice resulted in nodular glomerulosclerosis, mesangiolysis, microaneurysms, and arteriolar hyalinosis associated with massive proteinuria and renal failure in the absence of diabetic milieu or hypertension. In contrast, podocyte-specific VEGF(164) gain of function in wild-type mice resulted in less pronounced albuminuria and increased creatinine clearance. Transmission electron microscopy revealed glomerular basement membrane thickening and podocyte effacement in eNOS(-/-) mice with podocyte-specific VEGF(164) gain of function. Furthermore, glomerular nodules overexpressed collagen IV and laminin extensively. Biotin-switch and proximity ligation assays demonstrated that podocyte-specific VEGF(164) gain of function decreased glomerular S-nitrosylation of laminin in eNOS(-/-) mice. In addition, treatment with VEGF-A decreased S-nitrosylated laminin in cultured podocytes. Collectively, these data indicate that excess glomerular VEGF-A and eNOS deficiency is necessary and sufficient to induce Kimmelstiel-Wilson-like nodular glomerulosclerosis in mice through a process that involves deposition of laminin and collagen IV and de-nitrosylation of laminin.