Sharon C. Phillips

Role of the Gut Microbiome in Obstructive Sleep Apnea-Induced Hypertension.

Individuals suffering from obstructive sleep apnea (OSA) are at increased risk for systemic hypertension. The importance of a healthy gut microbiota, and detriment of a dysbiotic microbiota, on host physiology is becoming increasingly evident. We tested the hypothesis that gut dysbiosis contributes to hypertension observed with OSA. OSA was modeled in rats by inflating a tracheal balloon during the sleep cycle (10-s inflations, 60 per hour). On normal chow diet, OSA had no effect on blood pressure; however, in rats fed a high-fat diet, blood pressure increased 24 and 29 mm Hg after 7 and 14 days of OSA, respectively (P<0.05 each). Bacterial community characterization was performed on fecal pellets isolated before and after 14 days of OSA in chow and high-fat fed rats. High-fat diet and OSA led to significant alterations of the gut microbiota, including decreases in bacterial taxa known to produce the short chain fatty acid butyrate (P<0.05). Finally, transplant of dysbiotic cecal contents from hypertensive OSA rats on high-fat diet into OSA recipient rats on normal chow diet (shown to be normotensive) resulted in hypertension similar to that of the donor (increased 14 and 32 mm Hg after 7 and 14 days of OSA, respectively; P<0.05). These studies demonstrate a causal relationship between gut dysbiosis and hypertension, and suggest that manipulation of the microbiota may be a viable treatment for OSA-induced, and possibly other forms of, hypertension.

Intercellular Adhesion Molecule-1 Regulation In The Canine Lung After Cardiopulmonary Bypass

OBJECTIVE(S) Neutrophil sequestration in the lung after cardiopulmonary bypass has been shown to be dependent on the adhesion molecule CD18. Thus we sought to determine whether endothelial expression of intercellular adhesion molecule-1 (a ligand for CD18) in pulmonary capillaries mediates neutrophil adhesion in this setting. METHODS Seven adult mongrel dogs underwent 90 minutes of hypothermic cardiopulmonary bypass with 60 minutes of cardioplegic arrest. After warming, dogs were reperfused for up to 9 hours and lung biopsy specimens were obtained. Lung tissue was examined by Northern and Western blot analysis and by immunohistologic methods. Three sham-operated dogs served as time-matched controls. RESULTS Northern blots demonstrated increased expression of intercellular adhesion molecule-1 messenger ribonucleic acid within 5 minutes of cessation of bypass (or approximately 30 minutes after aortic crossclamp release), which persisted at 9 hours of recovery and was not present in controls. Western blots showed intercellular adhesion molecule-1 protein expression before bypass but a measurable increase in intercellular adhesion molecule-1 protein in four of seven dogs in the bypass group by the ninth hour of recovery. Pulmonary neutrophil accumulation 9 hours after cardiopulmonary bypass was greater in those dogs with an increased intercellular adhesion molecule-1 protein expression. Immunoelectron microscopy demonstrated the pulmonary capillary endothelium capable of increased intercellular adhesion molecule-1 protein expression at the 9-hour time point. CONCLUSIONS Cardiopulmonary bypass resulted in intercellular adhesion molecule-1 induction in the canine lung during recovery. An increased expression of intercellular adhesion molecule-1 protein in the lung was associated with an increased accumulation of neutrophils in affected animals. Thus intercellular adhesion molecule-1 expression may serve as a mechanism that predisposes the lungs to inflammatory cell-mediated injury postoperatively.

A new rodent model for obstructive sleep apnea: effects on ATP-mediated dilations in cerebral arteries

Obstructive sleep apnea (OSA), a condition in which the upper airway collapses during sleep, is strongly associated with metabolic and cardiovascular diseases. Little is known how OSA affects the cerebral circulation. The goals of this study were 1) to develop a rat model of chronic OSA that involved apnea and 2) to test the hypothesis that 4 wk of apneas during the sleep cycle alters endothelium-mediated dilations in middle cerebral arteries (MCAs). An obstruction device, which was chronically implanted into the trachea of rats, inflated to obstruct the airway 30 times/h for 8 h during the sleep cycle. After 4 wk of apneas, MCAs were isolated, pressurized, and exposed to luminally applied ATP, an endothelial P2Y2 receptor agonist that dilates through endothelial-derived nitric oxide (NO) and endothelial-dependent hyperpolarization (EDH). Dilations to ATP were attenuated ~30% in MCAs from rats undergoing apneas compared with those from a sham control group (P < 0.04 group effect; n = 7 and 10, respectively). When the NO component of the dilation was blocked to isolate the EDH component, the response to ATP in MCAs from the sham and apnea groups was similar. This finding suggests that the attenuated dilation to ATP must occur through reduced NO. In summary, we have successfully developed a novel rat model for chronic OSA that incorporates apnea during the sleep cycle. Using this model, we demonstrate that endothelial dysfunction occurred by 4 wk of apnea, likely increasing the vulnerability of the brain to cerebrovascular related accidents.

Pannexin Protein Expression in the Rat Middle Cerebral Artery

Background: Connexin proteins are well known to participate in cell-to-cell communication within the cerebral vasculature. Pannexins are a recently discovered family of proteins that could potentially be involved in cell-to-cell communication. Herein, we sought to determine whether pannexins are expressed in rat middle cerebral artery (MCA). Methods: A combination of RT-PCR, immunoblotting and immunohistochemistry techniques was used to characterize the expression pattern of pannexins in rat MCA. A fluorescent dye uptake approach in cultured smooth muscle cells was used to determine whether these cells have functional hemichannels. Results: We report for the first time that pannexins are expressed in the cerebral vasculature. We reveal that pannexin 1 is expressed in smooth muscle but not in endothelium and pannexin 2 is expressed in both endothelium and smooth muscle. Fluorescent dye entered cultured smooth muscle cells in the absence of extracellular calcium or when the cells were depolarized, which was prevented by the putative hemichannel blocker carbenoxolone. Conclusions: The identification of pannexins in rat MCA indicates that pannexin expression is not restricted to neuronal cells. Dye uptake in cultured smooth muscle cells exhibited properties similar to those of connexin and pannexin hemichannels, which may represent another form of cell-to-cell communication within the vasculature.

Disruption of K2P6.1 Produces Vascular Dysfunction and Hypertension in Mice

K2P6.1, a member of the 2-pore domain K channel family, is highly expressed in the vascular system; however, its function is unknown. We tested the following hypotheses. K2P6.1 regulates the following: (1) systemic blood pressure; (2) the contractile state of arteries; (3) vascular smooth muscle cell migration; (4) proliferation; and/or (5) volume regulation. Mice lacking K2P6.1 (KO) were generated by deleting exon 1 of Kcnk6. Mean arterial blood pressure in both anesthetized and awake KO mice was increased by 17±2 and 26±3 mm Hg, respectively (P<0.05). The resting membrane potential in freshly dispersed vascular smooth muscle cells was depolarized by 17±2 mV in the KO compared with wild-type littermates (P<0.05). The contractile responses to KCl (P<0.05) and BAY K 8644 (P<0.01), an activator of L-type calcium channels, were enhanced in isolated segments of aorta from KO mice. However, there was no difference in the current density of L-type calcium channels. Responses to U46619, an agent that activates rho kinase, showed an enhanced contraction in aorta from KO mice (P<0.001). The BAY K 8644-mediated increase in contraction was decreased to wild-type levels when treated with Y27632, a rho kinase inhibitor, (P<0.05). K2P6.1 does not appear to be involved with migration, proliferation, or volume regulation in cultured vascular smooth muscle cells. We conclude that K2P6.1 deficiency induces vascular dysfunction and hypertension through a mechanism that may involve smooth muscle cell depolarization and enhanced rho kinase activity.

Increased Cerebrovascular Sensitivity to Endothelin-1 in a Rat Model of Obstructive Sleep Apnea: A Role for Endothelin Receptor B:

Obstructive sleep apnea (OSA) is associated with cerebrovascular diseases. However, little is known regarding the effects of OSA on the cerebrovascular wall. We tested the hypothesis that OSA augments endothelin-1 (ET-1) constrictions of cerebral arteries. Repeated apneas (30 or 60 per hour) were produced in rats during the sleep cycle (8 hours) by remotely inflating a balloon implanted in the trachea. Four weeks of apneas produced a 23-fold increase in ET-1 sensitivity in isolated and pressurized posterior cerebral arteries (PCAs) compared with PCAs from sham-operated rats (EC50=10−9.2 mol/L versus 10−10.6 mol/L; P<0.001). This increased sensitivity was abolished by the ET-B receptor antagonist, BQ-788. Constrictions to the ET-B receptor agonist, IRL-1620, were greater in PCAs from rats after 2 or 4 weeks of apneas compared with that from sham-operated rats (P=0.013). Increased IRL-1620 constrictions in PCAs from OSA rats were normalized with the transient receptor potential channel (TRPC) blocker, SKF96365, or the Rho kinase (ROCK) inhibitor, Y27632. These data show that OSA increases the sensitivity of PCAs to ET-1 through enhanced ET-B activity, and enhanced activity of TRPCs and ROCK. We conclude that enhanced ET-1 signaling is part of a pathologic mechanism associated with adverse cerebrovascular outcomes of OSA.

Interleukin-6 Regulation In The Canine Myocardium After Cardiopulmonary Bypass ♦ 104

A systemic inflammatory reaction occurs in response to cardiopulmonary bypass (CPB). Recent evidence suggests myocardial involvement in this response. Interleukin-6 (IL-6) is a pleiotrophic cytokine with pro-inflammatory properties, known to induce cardiac myocyte intercellular adhesion molecule-1 (ICAM-1) production in vitro (thus supporting neutrophil-myocyte adhesion) and also known to have negative inotropic properties as well. Although plasma IL-6 levels have been shown to increase after CPB, the details of IL-6 induction and regulation after CPB are unknown. Also, tissue IL-6 levels may be physiologically more important than those in the circulation. Thus, to examine myocardial IL-6 regulation, dogs underwent 90 mins of hypothermic CPB (26-28°C) with 60 mins of cardioplegic arrest. After rewarming, dogs were reperfused open chest for either 3 hrs (n=4) or 6 hrs (n=4) at the end of which myocardial biopsies were obtained. An additional 4 dogs underwent open midline sternotomy without CPB and served as time-matched controls. Northern blot analysis of myocardial mRNA showed a marked increase in IL-6 in 3/4 dogs at 3 hrs and 3/4 dogs at 6 hrs of reperfusion when compared to sham bypass controls. Northern blots of mRNA extracted from isolated neutrophils and mononuclear leukocytes obtained from blood samples prior to CPB, at the end of CPB, and at 3 hrs of reperfusion failed to demonstrate IL-6 induction. In situ hybridization studies of the biopsied myocardium confirmed cardiac myocytes as a source of IL-6 production 3 hrs after CPB, with both sequestered monocytes and neutrophils also producing IL-6 by 6 hrs after CPB. Results of northern blots for ICAM-1 performed on the same myocardial tissue as the IL-6 studies showed ICAM-1 induction which was highly concordant with the IL-6 results. Thus, ischemia/reperfusion during CPB, despite myocardial protection with cold cardioplegic arrest, appears to be a sufficient stimulus to induce myocardial IL-6 synthesis. Myocardial IL-6 production may play a pivotal role in the development of depressed myocardial function postoperatively.

EVIDENCE FOR ADHESION MOLECULE AND CYTOKINE INDUCTION IN THE CANINE HEART FOLLOWING CARDIOPULMONARY BYPASS |[dagger]| 104

A systemic inflammatory reaction is known to occur in response to cardiopulmonary bypass (CPB). Recent data suggest leukocyte depletion after CPB may enhance myocardial function. To further investigate inflammation within the myocardium after CPB, northern blot analysis was performed for intercellular adhesion molecule-1 (ICAM-1), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-α) on tissue obtained from dogs undergoing 90 mins of hypothermic CPB (26-28°C) with 60 mins of cardioplegic arrest. After rewarming, dogs were reperfused open chest for 3, 6 or 9 hrs at which time myocardial samples were obtained. Animals undergoing open thoracotomy without CPB served as time-matched controls. Data are summarized in the table below. Positive results for IL-6 and ICAM-1 were concordant in those animals tested for both. IL-6 is known to be important in the induction of myocyte ICAM-1 in other models. ICAM-1 may serve as an adhesion ligand for infiltrating leukocytes. The presence of IL-8 and TNF-α suggest activated infiltrating leukocytes within the myocardium. These preliminary data indicate that inflammation after cardiopulmonary bypass does involve the myocardium. We speculate this inflammatory response may contribute to depressed myocardial function postoperatively.

INTERCELLULAR ADHESION MOLECULE-1 (ICAM-1) REGULATION IN THE CANINE LUNG AFTER CARDIOPULMONARY BYPASS (CPB). |[dagger]| 147

We have previously demonstrated a CD18-dependent sequestration of neutrophils in the dog lung within 3 hrs after CPB cessation that contributes to pulmonary dysfunction (Circulation 1995;92:2276-2283). To determine if expression of ICAM-1 (a ligand for CD18) on pulmonary capillary endothelium contributes to this response, 6 adult mongrel dogs underwent 90 mins of hypothermic CPB (26-28°C) with 60 mins of aortic cross-clamp time. After warming, dogs were reperfused open chest for 9 hrs. Lung biopsies were obtained prior to CPB, 10 to 30 mins after CPB, and at 1,2,3,6 and 9 hrs thereafter. Northern blots of lung biopsy homogenates demonstrated an early induction of the ICAM-1 gene which persisted at 9 hrs. In-situ hybridization showed mRNA expression within the alveolar wall, on alveolar macrophages, on bronchial epithelial cells, and on vascular endothelium of arterioles and venules. Western blots showed significant ICAM-1 expression prior to CPB, and against this background little or no increase in ICAM-1 protein expression was discernible after CPB. Immunohistochemistry performed at the light microscopic level confirmed ICAM-1 expression within the alveolar wall but failed to show an increase post-CPB, and failed to distinguish alveolar epithelial from capillary endothelial expression. Immunoelectron microscopy detected an increase in capillary ICAM-1 expression in only 1 dog at 9 hrs of reperfusion. Our data suggest that pulmonary neutrophil sequestration early after CPB, although CD18-dependent, is independent of pulmonary capillary ICAM-1 expression.