Nicotinamide nucleotide transhydrogenase activity impacts mitochondrial redox balance and the development of hypertension in mice.

Oxidant stress contributes to the initiation and progression of hypertension (HTN) by enhancing endothelial dysfunction and/or causing perturbations in nitric oxide homeostasis. Differences in mitochondrial function may augment this process and provide insight into why age of onset and clinical outcomes differ among individuals from distinct ethnic groups. We have previously demonstrated that variation in normal mitochondrial function and oxidant production exists in endothelial cells from individuals of Caucasian and African-American ethnicity and that this variation contributes to endothelial dysfunction. To model these distinct mitochondrial redox phenotypes, we used C57Bl/6N (6N) and C57Bl/6J (6J) mice that also display unique mitochondrial functional properties due to the differential expression nicotinamide nucleotide transhydrogenase (NNT). We demonstrate that the absence of NNT in 6J cells led to distinct mitochondrial bioenergetic profiles and a pro-oxidative mitochondrial phenotype characterized by increased superoxide production and reduced glutathione peroxidase activity. Interestingly, we found that 6J animals have significantly higher systolic blood pressure compared to 6N animals, and this difference is exacerbated by angiotensin II treatment. The changes in pressure were accompanied by both mitochondrial and vascular dysfunction revealed by impaired respiratory control ratios and endothelial-dependent vessel dilation. All end points could be significantly ameliorated by treatment with the mitochondria-targeted superoxide dismutase mimetic MitoTEMPO demonstrating a critical role for the production of mitochondrial reactive oxygen species in the development of HTN in these animals. Taken together, these data indicate that the absence of NNT leads to variation in mitochondrial function and contributes to a unique mitochondrial redox phenotype that influences susceptibility to HTN by contributing to endothelial and vascular dysfunction.

Pannexin 1 channels regulate leukocyte emigration through the venous endothelium during acute inflammation.

Inflammatory cell recruitment to local sites of tissue injury and/or infection is controlled by a plethora of signalling processes influencing cell-to-cell interactions between the vascular endothelial cells (ECs) in post-capillary venules and circulating leukocytes. Recently, ATP-sensitive P2Y purinergic receptors have emerged as downstream regulators of EC activation in vascular inflammation. However, the mechanism(s) regulating cellular ATP release in this response remains elusive. Here we report that the ATP-release channel Pannexin1 (Panx1) opens downstream of EC activation by TNF-α. This process involves activation of type-1 TNF receptors, recruitment of Src family kinases (SFK) and SFK-dependent phosphorylation of Panx1. Using an inducible, EC-specific Panx1 knockout mouse line, we report a previously unidentified role for Panx1 channels in promoting leukocyte adhesion and emigration through the venous wall during acute systemic inflammation, placing Panx1 channels at the centre of cytokine crosstalk with purinergic signalling in the endothelium.

Recruitment of the adaptor protein Nck to PECAM-1 couples oxidative stress to canonical NF-κB signaling and inflammation.

Oxidative stress stimulates nuclear factor κB (NF-κB) activation and NF-κB-dependent proinflammatory gene expression in endothelial cells during several pathological conditions, including ischemia/reperfusion injury. We found that the Nck family of adaptor proteins linked tyrosine kinase signaling to oxidative stress-induced activation of NF-κB through the classic IκB kinase-dependent pathway. Depletion of Nck prevented oxidative stress induced by exogenous hydrogen peroxide or hypoxia/reoxygenation injury from activating NF-κB in endothelial cells, increasing the abundance of the proinflammatory molecules ICAM-1 (intracellular adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1) and recruiting leukocytes. Nck depletion also attenuated endothelial cell expression of genes encoding proinflammatory factors but not those encoding antioxidants. Nck promoted oxidative stress-induced activation of NF-κB by coupling the tyrosine phosphorylation of PECAM-1 (platelet endothelial cell adhesion molecule-1) to the activation of p21-activated kinase, which mediates oxidative stress-induced NF-κB signaling. Consistent with this mechanism, treatment of mice subjected to ischemia/reperfusion injury in the cremaster muscle with a Nck inhibitory peptide blocked leukocyte adhesion and emigration and the accompanying vascular leak. Together, these data identify Nck as an important mediator of oxidative stress-induced inflammation and a potential therapeutic target for ischemia/reperfusion injury.

Platelets, acting in part via P-selectin, mediate cytomegalovirus-induced microvascular dysfunction.

Cytomegalovirus (CMV) infects a majority of the population worldwide. It has been implicated in cardiovascular disease, induces microvascular dysfunction, and synergizes with hypercholesterolemia to promote leukocyte and platelet recruitment in venules. Although platelets and platelet-associated P-selectin contribute to cardiovascular disease inflammation, their role in CMV-induced vascular responses is unknown. We assessed the role of platelets in CMV-induced microvascular dysfunction by depleting platelets and developing bone marrow chimeric mice deficient in platelet P-selectin. Wild-type and chimeric mice received mock or murine (m)CMV intraperitoneally. Five weeks later, some mice were switched to a high-cholesterol diet (HC) to investigate the synergism between mCMV and HC. Arteriolar vasodilation and recruitment of leukocytes and donor platelets in venules were measured at 11wk. mCMV with or without HC caused significant endothelial dysfunction in arterioles. Platelet depletion restored normal vasodilation in mCMV-HC but not mCMV-ND mice, whereas protection was seen in both groups for platelet P-selectin chimeras. Only mCMV + HC elevated leukocyte and platelet recruitment in venules. Leukocyte adhesion was reduced to mock levels by acute platelet depletion but was only partially decreased in platelet P-selectin chimeras. Platelets from mCMV-HC mice and, to a lesser extent, mCMV-ND but not mock-HC mice showed significant adhesion in mCMV-HC recipients. Our findings implicate a role for platelets, acting through P-selectin, in CMV-induced arteriolar dysfunction and suggest that the addition of HC leads to a platelet-dependent, inflammatory infiltrate that is only partly platelet P-selectin dependent. CMV appeared to have a stronger activating influence than HC on platelets and may represent an additional therapeutic target in vulnerable patients.

Silencing Bruton's tyrosine kinase in alveolar neutrophils protects mice from LPS/immune complex-induced acute lung injury.

Previous observations made by our laboratory indicate that Bruton's tyrosine kinase (Btk) may play an important role in the pathophysiology of local inflammation in acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). We have shown that there is cross talk between FcγRIIa and TLR4 in alveolar neutrophils from patients with ALI/ARDS and that Btk mediates the molecular cooperation between these two receptors. To study the function of Btk in vivo we have developed a unique two-hit model of ALI: LPS/immune complex (IC)-induced ALI. Furthermore, we conjugated F(ab)2 fragments of anti-neutrophil antibodies (Ly6G1A8) with specific siRNA for Btk to silence Btk specifically in alveolar neutrophils. It should be stressed that we are the first group to perform noninvasive transfections of neutrophils, both in vitro and in vivo. Importantly, our present findings indicate that silencing Btk in alveolar neutrophils has a dramatic protective effect in mice with LPS/IC-induced ALI, and that Btk regulates neutrophil survival and clearance of apoptotic neutrophils in this model. In conclusion, we put forward a hypothesis that Btk-targeted neutrophil specific therapy is a valid goal of research geared toward restoring homeostasis in lungs of patients with ALI/ARDS.

NAD(P)H oxidase and eNOS play differential roles in cytomegalovirus infection-induced microvascular dysfunction.

Primary cytomegalovirus (CMV) infection promotes oxidative stress and reduces nitric oxide (NO) bioavailability in endothelial cells. These events are among the earliest vascular responses to cardiovascular risk factors. We assessed the roles of NAD(P)H oxidase and NO bioavailability in microvascular responses to persistent CMV infection alone or with hypercholesterolemia. Wild-type (WT) or gp91(phox) (NAD(P)H oxidase subunit) knockout mice received mock inoculum or 3×10(4) PFU murine CMV (mCMV) ip 5 weeks before placement on a normal or high-cholesterol diet (HC) for 4 weeks before assessment of arteriolar function and venular blood cell recruitment using intravital microscopy. Some WT groups received sepiapterin (a precursor of the nitric oxide synthase cofactor tetrahydrobiopterin) or apocynin (NAD(P)H oxidase inhibitor/antioxidant). Endothelium-dependent vasodilation was impaired in mCMV vs mock WT, regardless of diet. This was not affected by sepiapterin, and pharmacological inhibition of nitric oxide synthase reduced dilation similarly in mock and mCMV mice. Apocynin or deficiency of total, but not blood cell or vascular wall only (tested using bone marrow chimeras), gp91(phox) protected against arteriolar dysfunction. Blood cell recruitment was induced by mCMV-HC. Sepiapterin, but not NAD(P)H oxidase deficiency/apocynin, reduced leukocyte accumulation, whereas platelet adhesion was reduced by sepiapterin, apocynin, or total, platelet-specific, or vascular wall gp91(phox) deficiency. These data implicate activation of both hematopoietic and vessel wall NAD(P)H oxidase in mCMV-induced arteriolar dysfunction and platelet and vascular NAD(P)H oxidase in the thrombogenic phenotype induced by mCMV-HC. In contrast, findings with sepiapterin suggest that eNOS dysfunction, perhaps uncoupling, mediates venular, but not arteriolar, responses to mCMV-HC, thus indicating that NAD(P)H oxidase and eNOS differentially regulate microvascular responses to mCMV.

P-selectin mediates the microvascular dysfunction associated with persistent cytomegalovirus infection in normocholesterolemic and hypercholesterolemic mice.

OBJECTIVE: Cytomegalovirus has been implicated in cardiovascular disease, possibly through the induction of inflammatory processes. P-selectin and L-selectin are adhesion molecules that mediate early microvascular responses to inflammatory stimuli. This study examined the role of these selectins in the microvascular dysfunction that occurs during persistent CMV infection. METHODS: C57Bl/6, P- or L-selectin-deficient mice were mock-inoculated or infected with murine CMV, and five weeks later placed on normal diet or high cholesterol diet for six weeks. P-selectin expression was measured or intravital microscopy was performed to determine arteriolar vasodilation and venular blood cell recruitment. RESULTS: P-selectin expression was significantly increased in the heart, lung, and spleen of mCMV-ND, but not mCMV-HC C57Bl/6. mCMV-ND and mCMV-HC exhibited impaired arteriolar function, which was reversed by treatment with an anti-P-selectin antibody, but not L-selectin deficiency. mCMV-HC also showed elevated leukocyte and platelet recruitment. P-selectin inhibition abrogated, whereas L-selectin deficiency partially reduced these responses. CONCLUSIONS: We provide the first evidence for P-selectin upregulation by persistent mCMV infection and implicate this adhesion molecule in the associated arteriolar dysfunction. P-selectin, and to a lesser extent L-selectin, mediates the leukocyte and platelet recruitment induced by CMV infection combined with hypercholesterolemia.

Cytomegalovirus infection leads to microvascular dysfunction and exacerbates hypercholesterolemia-induced responses.

Cytomegalovirus (CMV) persistently infects more than 60% of the worldwide population. In immunocompetent hosts, it has been implicated in several diseases, including cardiovascular disease, possibly through the induction of inflammatory pathways. Cardiovascular risk factors promote an inflammatory phenotype in the microvasculature long before clinical disease is evident. This study determined whether CMV also impairs microvascular homeostasis and synergizes with hypercholesterolemia to exaggerate these responses. Intravital microscopy was used to assess endothelium-dependent and -independent arteriolar vasodilation and venular leukocyte and platelet adhesion in mice after injection with either mock inoculum or murine CMV (mCMV). Mice were fed a normal (ND) or high-cholesterol (HC) diet beginning at 5 weeks postinfection (p.i.), or a HC diet for the final 4 weeks of infection. mCMV-ND mice exhibited impaired endothelium-dependent vasodilation versus mock-ND at 9 and 12 weeks and endothelium-independent arteriolar dysfunction by 24 weeks. Transient mild leukocyte adhesion occurred in mCMV-ND venules at 7 and 21 weeks p.i. HC alone caused temporary arteriolar dysfunction and venular leukocyte and platelet recruitment, which were exaggerated and prolonged by mCMV infection. The time of introduction of HC after mCMV infection determined whether mCMV+HC led to worse venular inflammation than either factor alone. These findings reveal a proinflammatory influence of persistent mCMV on the microvasculature, and suggest that mCMV infection enhances microvasculature susceptibility to both inflammatory and thrombogenic responses caused by hypercholesterolemia.

Effects of chronic and acute stressors and CRF on depression-like behavior in mice.

The effects of chronic footshock (CFS) on behavioral responses of CD1 mice to acute footshock and restraint were studied in tests commonly used to assess antidepressant treatments. Adult male mice were subjected to 20 min of footshock daily for 14-16 days, and then tested in the tail suspension test (TST) and the forced swim test (FST). CFS treatment did not alter immobility in the TST when mice were tested before the footshock on that day. However, when the TST was performed after the footshock, immobility decreased in both control and CFS mice. In the FST, chronic footshock significantly increased the time spent floating when mice were tested before footshock on that day. However, when the FST was performed immediately after the footshock, floating decreased in the CFS mice, but not in previously unshocked mice. Restraint, shortly before the FST, decreased floating in both CFS and unshocked mice. Thus, CFS induced depression-like activity in the FST, but not in the TST, whereas acute footshock or restraint immediately before testing induced antidepressant-like effects in both the TST and the FST. In unshocked mice, intracerebroventricular corticotropin-releasing factor (CRF) consistently decreased immobility in the TST and the FST, with significant effects at the 100ng dose. The same dose of CRF depressed activity in the open field, so that these changes in immobility are unlikely to reflect a change in overall activity. CRF thus mimicked the effects of the acute stressors in the TST and the FST. Responses to icv CRF were attenuated by chronic footshock suggesting that CFS desensitizes the brain to CRF. CFS treatment did not alter basal concentrations of ACTH and corticosterone in blood plasma. Acute footshock increased the plasma concentrations of the hormones but in CFS mice these responses were attenuated, significantly for plasma ACTH. Acute footshock activated brain dopamine, norepinephrine and serotonin metabolism, and increased tryptophan concentrations in the brain. In CFS mice, these responses were attenuated, significantly for hypothalamic NE.