The gut microbiome contributes to blood-brain barrier disruption in spontaneously hypertensive stroke prone rats.

In recent years, it has become apparent that the gut microbiome can influence the functioning and pathological states of organs and systems throughout the body. In this study, we tested the hypothesis that the gut microbiome has a major role in the disruption of the blood-brain barrier (BBB) in the spontaneously hypertensive stroke prone rats (SHRSP), an animal model for hypertensive cerebral small vessel disease (CSVD). Loss of BBB is thought to be an early and initiating component to the full expression of CSVD in animal models and humans. To test this hypothesis, newly born SHRSP pups were placed with foster dams of the SHRSP strain or dams of the WKY strain, the control strain that does not demonstrate BBB dysfunction or develop hypertensive CSVD. Similarly, WKY pups were placed with foster dams of the same or opposite strain. The rationale for cross fostering is that the gut microbiomes are shaped by environmental bacteria of the foster dam and the nesting surroundings. Analysis of the bacterial genera in feces, using 16S rRNA analysis, demonstrated that the gut microbiome in the rat pups was influenced by the foster dam. SHRSP offspring fostered on WKY dams had systolic blood pressures (SBPs) that were significantly decreased by 26 mmHg (P < .001) from 16-20 weeks, compared to SHRSP offspring fostered on SHRSP dams. Similarly WKY offspring fostered on SHRSP dams had significantly increased SBP compared to WKY offspring fostered on WKY dams, although the magnitude of SBP change was not as robust. At ~20 weeks of age, rats fostered on SHRSP dams showed enhanced inflammation in distal ileum regardless of the strain of the offspring. Disruption of BBB integrity, an early marker of CSVD onset, was improved in SHRSPs that were fostered on WKY dams when compared to the SHRSP rats fostered on SHRSP dams. Although SHRSP is a genetic model for CSVD, environmental factors such as the gut microbiota of the foster dam have a major influence in the loss of BBB integrity.

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.

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.

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 K(2P)6.1 produces vascular dysfunction and hypertension in mice.

K(2P)6.1, 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. K(2P)6.1 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 K(2P)6.1 (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). K(2P)6.1 does not appear to be involved with migration, proliferation, or volume regulation in cultured vascular smooth muscle cells. We conclude that K(2P)6.1 deficiency induces vascular dysfunction and hypertension through a mechanism that may involve smooth muscle cell depolarization and enhanced rho kinase activity.

Evidence for two-pore domain potassium channels in rat cerebral arteries.

Little is known about the presence and function of two-pore domain K(+) (K(2P)) channels in vascular smooth muscle cells (VSMCs). Five members of the K(2P) channel family are known to be directly activated by arachidonic acid (AA). The purpose of this study was to determine 1) whether AA-sensitive K(2P) channels are expressed in cerebral VSMCs and 2) whether AA dilates the rat middle cerebral artery (MCA) by increasing K+ currents in VSMCs via an atypical K+ channel. RT-PCR revealed message for the following AA-sensitive K(2P) channels in rat MCA: tandem of P domains in weak inward rectifier K+ (TWIK-2), TWIK-related K+ (TREK-1 and TREK-2), TWIK-related AA-stimulated K+ (TRAAK), and TWIK-related halothane-inhibited K+ (THIK-1) channels. However, in isolated VSMCs, only message for TWIK-2 was found. Western blotting showed that TWIK-2 is present in MCA, and immunohistochemistry further demonstrated its presence in VSMCs. AA (10-100 microM) dilated MCAs through an endothelium-independent mechanism. AA-induced dilation was not affected by inhibition of cyclooxygenase, epoxygenase, or lipoxygenase or inhibition of classical K+ channels with 10 mM TEA, 3 mM 4-aminopyridine, 10 microM glibenclamide, or 100 microM Ba2+. AA-induced dilations were blocked by 50 mM K+, indicating involvement of a K+ channel. AA (10 microM) increased whole cell K+ currents in dispersed cerebral VSMCs. AA-induced currents were not affected by inhibitors of the AA metabolic pathways or blockade of classical K+ channels. We conclude that AA dilates the rat MCA and increases K+ currents in VSMCs via an atypical K+ channel that is likely a member of the K(2P) channel family.