A 3D biomimetic model of lymphatics reveals cell-cell junction tightening and lymphedema via a cytokine-induced ROCK2/JAM-A complex.

Impaired lymphatic drainage and lymphedema are major morbidities whose mechanisms have remained obscure. To study lymphatic drainage and its impairment, we engineered a microfluidic culture model of lymphatic vessels draining interstitial fluid. This lymphatic drainage-on-chip revealed that inflammatory cytokines that are known to disrupt blood vessel junctions instead tightened lymphatic cell-cell junctions and impeded lymphatic drainage. This opposing response was further demonstrated when inhibition of rho-associated protein kinase (ROCK) was found to normalize fluid drainage under cytokine challenge by simultaneously loosening lymphatic junctions and tightening blood vessel junctions. Studies also revealed a previously undescribed shift in ROCK isoforms in lymphatic endothelial cells, wherein a ROCK2/junctional adhesion molecule-A (JAM-A) complex emerges that is responsible for the cytokine-induced lymphatic junction zippering. To validate these in vitro findings, we further demonstrated in a genetic mouse model that lymphatic-specific knockout of ROCK2 reversed lymphedema in vivo. These studies provide a unique platform to generate interstitial fluid pressure and measure the drainage of interstitial fluid into lymphatics and reveal a previously unappreciated ROCK2-mediated mechanism in regulating lymphatic drainage.

Vascular smooth muscle cell c-Fos is critical for foam cell formation and atherosclerosis.

BACKGROUND: Hyperlipidemia-induced vascular smooth muscle cell (VSMC)-derived foam cell formation is considered a crucial event in the development of atherosclerosis. Since c-Fos emerges as a key modulator of lipid metabolism, we investigated whether c-Fos plays a role in hyperlipidemia-induced VSMC-derived foam cell formation and atherosclerosis. APPROACH AND RESULTS: c-Fos expression was observed in VSMCs in atherosclerotic plaques from patients and western diet-fed atherosclerosis-prone LDLR-/- and ApoE-/- mice by immunofluorescence staining. To ascertain c-Fos's function in atherosclerosis development, VSMC-specific c-Fos deficient mice in ApoE-/- background were established. Western diet-fed c-FosVSMCKOApoE-/- mice exhibited a significant reduction of atherosclerotic lesion formation as measured by hematoxylin and eosin staining, accompanied by decreased lipid deposition within aortic roots as determined by Oil red O staining. Primary rat VSMCs were isolated to examine the role of c-Fos in lipid uptake and foam cell formation. oxLDL stimulation resulted in VSMC-derived foam cell formation and elevated intracellular mitochondrial reactive oxygen species (mtROS), c-Fos and LOX-1 levels, whereas specific inhibition of mtROS, c-Fos or LOX-1 lessened lipid accumulation in oxLDL-stimulated VSMCs. Mechanistically, oxLDL acts through mtROS to enhance transcription activity of c-Fos to facilitate the expression of LOX-1, exerting a feedforward mechanism with oxLDL to increase lipid uptake and propel VSMC-derived foam cell formation and atherogenesis. CONCLUSION: Our study demonstrates a fundamental role of mtROS/c-Fos/LOX-1 signaling pathway in promoting oxLDL uptake and VSMC-derived foam cell formation during atherosclerosis. c-Fos may represent a promising therapeutic target amenable to clinical translation in the future.

Diabetes and Its Cardiovascular Complications: Comprehensive Network and Systematic Analyses.

Diabetes mellitus is a worldwide health problem that usually comes with severe complications. There is no cure for diabetes yet and the threat of these complications is what keeps researchers investigating mechanisms and treatments for diabetes mellitus. Due to advancements in genomics, epigenomics, proteomics, and single-cell multiomics research, considerable progress has been made toward understanding the mechanisms of diabetes mellitus. In addition, investigation of the association between diabetes and other physiological systems revealed potentially novel pathways and targets involved in the initiation and progress of diabetes. This review focuses on current advancements in studying the mechanisms of diabetes by using genomic, epigenomic, proteomic, and single-cell multiomic analysis methods. It will also focus on recent findings pertaining to the relationship between diabetes and other biological processes, and new findings on the contribution of diabetes to several pathological conditions.

Non-alcoholic Steatohepatitis Pathogenesis, Diagnosis, and Treatment.

There has been a rise in the prevalence of non-alcohol fatty liver disease (NAFLD) due to the popularity of western diets and sedentary lifestyles. One quarter of NAFLD patients is diagnosed with non-alcoholic steatohepatitis (NASH), with histological evidence not only of fat accumulation in hepatocytes but also of liver cell injury and death due to long-term inflammation. Severe NASH patients have increased risks of cirrhosis and liver cancer. In this review, we discuss the pathogenesis and current methods of diagnosis for NASH, and current status of drug development for this life-threatening liver disease.

Endocytic Adaptors in Cardiovascular Disease.

Endocytosis is the process of actively transporting materials into a cell by membrane engulfment. Traditionally, endocytosis was divided into three forms: phagocytosis (cell eating), pinocytosis (cell drinking), and the more selective receptor-mediated endocytosis (clathrin-mediated endocytosis); however, other important endocytic pathways (e.g., caveolin-dependent endocytosis) contribute to the uptake of extracellular substances. In each, the plasma membrane changes shape to allow the ingestion and internalization of materials, resulting in the formation of an intracellular vesicle. While receptor-mediated endocytosis remains the best understood pathway, mammalian cells utilize each form of endocytosis to respond to their environment. Receptor-mediated endocytosis permits the internalization of cell surface receptors and their ligands through a complex membrane invagination process that is facilitated by clathrin and adaptor proteins. Internalized vesicles containing these receptor-ligand cargoes fuse with early endosomes, which can then be recycled back to the plasma membrane, delivered to other cellular compartments, or destined for degradation by fusing with lysosomes. These intracellular fates are largely determined by the interaction of specific cargoes with adaptor proteins, such as the epsins, disabled-homolog 2 (Dab2), the stonin proteins, epidermal growth factor receptor substrate 15, and adaptor protein 2 (AP-2). In this review, we focus on the role of epsins and Dab2 in controlling these sorting processes in the context of cardiovascular disease. In particular, we will focus on the function of epsins and Dab2 in inflammation, cholesterol metabolism, and their fundamental contribution to atherogenicity.

Impact of Acute and Chronic Hypertension on Changes in Pial Collateral Tone In Vivo During Transient Ischemia.

We investigated vasoconstrictive responses of pial collaterals in vivo at baseline and during transient middle cerebral artery occlusion during chronic hypertension. A cranial window was used to measure diameter of leptomeningeal anastomoses (pial collaterals) in male Wistar (n=8) and spontaneously hypertensive rats (SHRs; n=8) using video dimensional analysis. Middle cerebral artery occlusion was induced by remote filament for 2 hours with 2 hours reperfusion. Phenylephrine was infused during ischemia as a pressor therapy. Active diameters of pial collaterals were significantly smaller in SHRs versus Wistar (14.1±1.5 versus 21.6±2.8 µm; P<0.01); however, passive diameters were similar (25.0±2.9 versus 25.0±2.6 µm; P>0.05). Basal tone of pial collaterals before occlusion was 42±5% in SHRs versus 15±4% in Wistar (P<0.01). Tone decreased in both Wistar and SHRs during occlusion but remained higher in SHRs (9±2% versus 29±4%; P<0.05). Phenylephrine increased blood pressure in both groups but had little effect on leptomeningeal anastomoses diameters. Reperfusion caused vasoconstriction of pial collaterals, increasing tone from 8±1% to 20±5% in Wistar and 29±5% to 44±5% in SHRs (P<0.01). Higher tone in pial collaterals from SHRs basally and during occlusion/reperfusion could limit flow to the penumbra and promote evolution of infarction. Sustained elevated tone of pial collaterals from SHRs with phenylephrine suggests pressor therapy may not be appropriate during chronic hypertension.

Transient receptor potential vanilloid-4 channels are involved in diminished myogenic tone in brain parenchymal arterioles in response to chronic hypoperfusion in mice.

AIM: Adaptive responses of brain parenchymal arterioles (PAs), a target for cerebral small vessel disease, to chronic cerebral hypoperfusion are largely unknown. Previous evidence suggested that transient receptor potential vanilloid 4 channels may be involved in the regulation of cerebrovascular tone. Therefore, we investigated the role of TRPV4 in adaptations of PAs in a mouse model of chronic hypoperfusion. METHODS: TRPV4 knockout (-/- ) and wild-type (WT) mice were subjected to unilateral common carotid artery occlusion (UCCAo) for 28 days. Function and structure of PAs ipsilateral to UCCAo were studied isolated and pressurized in an arteriograph. RESULTS: Basal tone of PAs was similar between WT and TRPV4-/- mice (22 ± 3 vs 23 ± 5%). After UCCAo, active inner diameters of PAs from WT mice were larger than control (41 ± 2 vs 26 ± 5 µm, P < 0.05) that was due to decreased tone (8 ± 2 vs 23 ± 5%, P < 0.05), increased passive inner diameters (46 ± 3 vs 34 ± 2 µm, P < 0.05), and decreased wall-to-lumen ratio (0.104 ± 0.01 vs 0.137 ± 0.01, P < 0.05). However, UCCAo did not affect vasodilation to a small- and intermediate-conductance calcium-activated potassium channel agonist NS309, the nitric oxide (NO) donor sodium nitroprusside, or constriction to a NO synthase inhibitor L-NNA. Wall thickness and distensibility in PAs from WT mice were unaffected. In TRPV4-/- mice, UCCAo had no effect on active inner diameters or tone and only increased passive inner diameters (53 ± 2 vs 43 ± 3 µm, P < 0.05). CONCLUSION: Adaptive response of PAs to chronic cerebral hypoperfusion includes myogenic tone reduction and outward remodelling. TRPV4 channels were involved in tone reduction but not outward remodelling in response to UCCAo.

The importance of comorbidities in ischemic stroke: Impact of hypertension on the cerebral circulation.

Comorbidities are a hallmark of stroke that both increase the incidence of stroke and worsen outcome. Hypertension is prevalent in the stroke population and the most important modifiable risk factor for stroke. Hypertensive disorders promote stroke through increased shear stress, endothelial dysfunction, and large artery stiffness that transmits pulsatile flow to the cerebral microcirculation. Hypertension also promotes cerebral small vessel disease through several mechanisms, including hypoperfusion, diminished autoregulatory capacity and localized increase in blood-brain barrier permeability. Preeclampsia, a hypertensive disorder of pregnancy, also increases the risk of stroke 4-5-fold compared to normal pregnancy that predisposes women to early-onset cognitive impairment. In this review, we highlight how comorbidities and concomitant disorders are not only risk factors for ischemic stroke, but alter the response to acute ischemia. We focus on hypertension as a comorbidity and its effects on the cerebral circulation that alters the pathophysiology of ischemic stroke and should be considered in guiding future therapeutic strategies.

Inhibition of PAI (Plasminogen Activator Inhibitor)-1 Improves Brain Collateral Perfusion and Injury After Acute Ischemic Stroke in Aged Hypertensive Rats.

Background and Purpose- Aging and hypertension, comorbidities prevalent in the stroke population, are associated with poor collateral status and worsened stroke outcome. However, underlying mechanisms by which these conditions affect stroke outcome are not clear. We studied the role of PAI (plasminogen activator inhibitor)-1 that is increased in aging and hypertension on brain and vascular expression of inflammatory factors and perfusion that may contribute to worse stroke outcomes. Methods- Aged (≈50 weeks) and young (≈18 weeks) spontaneously hypertensive rats (SHR) were subjected to ischemia by middle cerebral artery occlusion (2 hours) and reperfusion (2 hours) with or without treatment with the PAI-1 inhibitor TM5441. Changes in middle cerebral artery and collateral perfusion territories were measured by multisite laser Doppler. Reactivity to TM5441 was studied using isolated and pressurized leptomeningeal anastomotic arterioles. Brain injury was determined by 2,3,5-triphenyltetrazolium staining and quantitative immunohistochemistry of amyloid-ß-42, PAI-1, and hemoglobin. Circulating inflammatory factors were measured by ELISA. Results- Changes in cerebral blood flow during middle cerebral artery occlusion were similar between groups, with both having poor collateral perfusion and incomplete reperfusion. However, aged SHR had greater brain injury versus young (41±2 versus 23±2%, P<0.05) as well as increased brain deposition of amyloid-ß-42 and circulating oxLDL (oxidized low-density lipoprotein). Erythrocyte aggregation and hemorrhage within the injured brain was observed in 50% of aged but no young SHR, with increased circulating PAI-1 in this subgroup of aged SHR (16±3 versus 6±2 ng/mL, P<0.05). PAI-1 inhibition with TM5441 improved brain injury but did not affect hemorrhage. TM5441 increased collateral perfusion by 38±7% and dilated leptomeningeal anastomotic arterioles by 44±10%, which was abolished by nitric oxide synthase inhibition. Conclusions- Increased injury in aged SHR seemed to be related to poor collateral perfusion, hemorrhagic transformation, increased amyloid-ß-42, and oxidative stress. PAI-1 inhibition reduced infarction in both groups of SHR that possibly due, in part, to increased collateral perfusion.

Effect of TTC Treatment on Immunohistochemical Quantification of Collagen IV in Rat Brains after Stroke.

Although used extensively in stroke research, there is limited knowledge of how 2, 3, 5-triphenyltetrazolium chloride (TTC)-treated rat brain sections are altered and if they can be used for immunohistochemical quantification after staining with TTC. In the present study, we hypothesized that TTC treatment (TTC+) would not interfere with collagen IV immunohistochemical staining compared with non-TTC-treated (TTC-) brain slices. We further hypothesized that there would be no difference in autofluorescence or nonspecific secondary antibody fluorescence between TTC+ and TTC- brain slices. Coronal brain sections of male Wistar rats (n = 5/group) were either treated with TTC or not after middle cerebral artery occlusion or sham surgery, and processed for immunohistochemical staining with mouse anti-collagen IV as the primary antibody, and goat anti-IgM as the secondary antibody. Four images were taken in the cerebral cortex of the contralateral side of infarction in each brain slice using an Olympus BX50 fluorescence microscope, and average intensity of the entire image was quantified using the Metamorph software. Compared with TTC- brain slices, TTC+ brain slices showed a significantly lower autofluorescence (P < 0.05), but was unchanged for nonspecific secondary antibody fluorescence. In addition, TTC+ brain slices had similar collagen IV staining intensity compared with TTC- brain slices. These results demonstrate that TTC+ brain slices are usable for immunohistochemical quantification.

Pharmacologically increasing collateral perfusion during acute stroke using a carboxyhemoglobin gas transfer agent (Sanguinate™) in spontaneously hypertensive rats.

Similar to patients with chronic hypertension, spontaneously hypertensive rats (SHR) develop fast core progression during middle cerebral artery occlusion (MCAO) resulting in large final infarct volumes. We investigated the effect of Sanguinate™ (SG), a PEGylated carboxyhemoglobin (COHb) gas transfer agent, on changes in collateral and reperfusion cerebral blood flow and brain injury in SHR during 2 h of MCAO. SG (8 mL/kg) or vehicle ( n = 6-8/group) was infused i.v. after 30 or 90 min of ischemia with 2 h reperfusion. Multi-site laser Doppler probes simultaneously measured changes in core MCA and collateral flow during ischemia and reperfusion using a validated method. Brain injury was measured using TTC. Animals were anesthetized with choral hydrate. Collateral flow changed little in vehicle-treated SHR during ischemia (-8 ± 9% vs. prior to infusion) whereas flow increased in SG-treated animals (29 ± 10%; p < 0.05). In addition, SG improved reperfusion regardless of time of treatment; however, brain injury was smaller only with early treatment in SHR vs. vehicle (28.8 ± 3.2% vs. 18.8 ± 2.3%; p < 0.05). Limited collateral flow in SHR during MCAO is consistent with small penumbra and large infarction. The ability to increase collateral flow in SHR with SG suggests that this compound may be useful as an adjunct to endovascular therapy and extend the time window for treatment.

Treatment with low dose fasudil for acute ischemic stroke in chronic hypertension.

We investigated the effect of Rho kinase inhibition on changes in cerebral blood flow (CBF), brain injury and vascular function after ischemic stroke in spontaneously hypertensive rats (SHR). Changes in core MCA and collateral perfusion were measured by a validated laser Doppler method. Animals underwent 2 h tMCAO and 2 h reperfusion. Fasudil (0.1 mg/kg, i.v.) or vehicle was given at 30 min ischemia (n = 9/group; mean (SD)). Brain injury was determined by 2,3,5-triphenyltetrazolium chloride staining. To determine the effect of fasudil on vascular function, fasudil was given 10 min before reperfusion and parenchymal arterioles studied isolated (n = 6/group; mean(SD)). Collateral perfusion was low in vehicle-treated SHR (-8(32)%) that changed minimally with fasudil (6(24)%, p > 0.05, effect size: 0.47;95% CI-0.49-1.39). Reperfusion CBF was below baseline in vehicle (-27(26)%) and fasudil (-32(25)%, p > 0.05, effect size: 0.19; 95% CI-0.74-1.11) groups, suggesting incomplete reperfusion in both groups. Fasudil had little effect on brain injury volume (28(13)% vs. 36(7)% in vehicle, p > 0.05, effect size: 0.75; 95% CI-0.24-1.66). In isolated parenchymal arterioles, myogenic tone was similar between groups (37(6)% vs. 38(10)% in vehicle, p > 0.05, effect size: 0.09; 95% CI-1.05-1.21). There were no differences with fasudil treatment vs. vehicle in perfusion, brain injury and vascular function that may be related to the low dose that had minimal blood pressure lowering effect.

Effect of hypertension and peroxynitrite decomposition with FeTMPyP on CBF and stroke outcome.

We investigated the effect of peroxynitrite decomposition catalyst FeTMPyP treatment on perfusion deficit, vascular function and stroke outcome in Wistar ( n = 26) and spontaneously hypertensive rats stroke-prone (SHRSP; n = 26) that underwent tMCAO for 2 h or Sham operation. Peri-infarct CBF was measured by hydrogen clearance in the absence or presence of FeTMPyP (10 mg/kg, i.v.) or vehicle 10 min before reperfusion. Myogenic tone of parenchymal arterioles (PAs) was measured as an indication of small vessel resistance (SVR). Baseline CBF was similar between Wistar and SHRSP (114 ± 12 vs. 132 ± 9 mL/100 g/min); however, MCAO caused greater perfusion deficit in SHRSP (24 ± 6 vs. 7 ± 1 mL/100 g/min; p < 0.05) and increased infarct volume by TTC (12 ± 6 vs. 32 ± 2%; p < 0.05). Reperfusion CBF was decreased from baseline in both SHRSP and Wistar (54 ± 16 and 46 ± 19 mL/100 g/min; p < 0.05), suggesting increased infarction in SHRSP was related to greater perfusion deficit. PAs from SHRSP had increased tone vs. Wistar that was enhanced after tMCAO. FeTMPyP treatment did not affect CBF during ischemia or reperfusion, or tone of PAs, but decreased the incidence of hemorrhage in SHRSP by 50%. Thus, increased tone in PAs from SHRSP could increase perfusion deficit during MCAO that was not alleviated by FeTMPyP.

Pial Collateral Reactivity During Hypertension and Aging: Understanding the Function of Collaterals for Stroke Therapy.

BACKGROUND AND PURPOSE: We investigated vasoactive properties of leptomeningeal arterioles (LMAs) under normotensive conditions and during hypertension and aging that are known to have poor collateral flow and little salvageable tissue. METHODS: LMAs, identified as distal anastomotic arterioles connecting middle and anterior cerebral arteries, were studied isolated and pressurized from young (18 weeks) or aged (48 weeks) normotensive Wistar Kyoto (WKY18, n=14; WKY48, n=6) rats and spontaneously hypertensive rats (SHR18, n=16; SHR48, n=6). Myogenic tone and vasoactive responses to pressure as well as endothelial function and ion channel activity were measured. RESULTS: LMAs from WKY18 had little myogenic tone at 40 mm Hg (8±3%) that increased in aged WKY48 (30±6%). However, LMAs from both WKY groups dilated to increased pressure and demonstrated little myogenic reactivity, a response that would be conducive to collateral flow. In contrast, LMAs from both SHR18 and SHR48 displayed considerable myogenic tone (56±8% and 43±7%; P<0.01 versus WKY) and constricted to increased pressure. LMAs from both WKY and SHR groups had similar basal endothelial nitric oxide and IK channel activity that opposed tone. However, dilation to sodium nitroprusside, diltiazem and 15 mmol/L KCl was impaired in LMAs from SHR18. CONCLUSIONS: This study shows for the first time that LMAs from young and aged SHR are vasoconstricted and have impaired vasodilatory responses that may contribute to greater perfusion deficit and little penumbral tissue. These results also suggest that therapeutic opening of pial collaterals is possible during middle cerebral artery occlusion to create penumbral tissue and prevent infarct expansion.

Roles of Caveolin-1 in Angiotensin II-Induced Hypertrophy and Inward Remodeling of Cerebral Pial Arterioles.

Angiotensin II (Ang II) is a major determinant of inward remodeling and hypertrophy in pial arterioles that may have an important role in stroke during chronic hypertension. Previously, we found that epidermal growth factor receptor is critical in Ang II-mediated hypertrophy that may involve caveolin-1 (Cav-1). In this study, we examined the effects of Cav-1 and matrix metalloproteinase-9 (MMP9) on Ang II-mediated structural changes in pial arterioles. Cav-1-deficient (Cav-1(-/-)), MMP9-deficient (MMP9(-/-)), and wild-type mice were infused with either Ang II (1000 ng/kg per minute) or saline via osmotic minipumps for 28 days (n=6-8 per group). Systolic arterial pressure was measured by a tail-cuff method. Pressure and diameter of pial arterioles were measured through an open cranial window in anesthetized mice. Cross-sectional area of the wall was determined histologically in pressurized fixed pial arterioles. Expression of Cav-1, MMP9, phosphorylated epidermal growth factor receptor, and Akt was determined by Western blotting and immunohistochemistry. Deficiency of Cav-1 or MMP9 did not affect Ang II-induced hypertension. Ang II increased the expression of Cav-1, phosphorylated epidermal growth factor receptor, and Akt in wild-type mice, which was attenuated in Cav-1(-/-) mice. Ang II-induced hypertrophy, inward remodeling, and increased MMP9 expression in pial arterioles were prevented in Cav-1(-/-) mice. Ang II-mediated increases in MMP9 expression and inward remodeling, but not hypertrophy, were prevented in MMP9(-/-) mice. In conclusion, Cav-1 is essential in Ang II-mediated inward remodeling and hypertrophy in pial arterioles. Cav-1-induced MMP9 is exclusively involved in inward remodeling, not hypertrophy. Further studies are needed to determine the role of Akt in Ang II-mediated hypertrophy.

Effect of hypertension and carotid occlusion on brain parenchymal arteriole structure and reactivity.

We studied the effect of hypertension and chronic hypoperfusion on brain parenchymal arteriole (PA) structure and function. PAs were studied isolated and pressurized from 18-wk-old Wistar-Kyoto (WKY18; n = 8) and spontaneously hypertensive stroke prone (SHRSP18; n = 8) and 5-wk-old prehypertensive (SHRSP5; n = 8) rats. In separate groups, unilateral common carotid artery occlusion (UCCAo) was performed for 4 wk to cause chronic hypoperfusion in 18-wk-old WKY (WKY18-CH; n = 8) and SHRSP (SHRSP18-CH; n = 8). UCCAo caused PAs to have significantly diminished myogenic tone (31 ± 3 vs. 14 ± 6% at 60 mmHg; P < 0.05) and reactivity to pressure from WKY18-CH vs. WKY18 animals. The effect of UCCAo was limited to normotensive animals, as there was little effect of chronic hypoperfusion on vascular reactivity or percent tone in PAs from SHRSP18 vs. SHRSP18-CH animals (53 ± 4 vs. 41 ± 3%; P > 0.05). However, PAs from SHRSP18 and SHRSP5 animals had significantly greater tone compared with WKY18, suggesting an effect of strain and not hypertension per se on PA vasoconstriction. Structurally, PAs from SHRSP18 and SHRSP5 animals had similar sized lumen diameters, but increased wall thickness and distensibility compared with WKY18. Interestingly, chronic hypoperfusion did not affect the structure of PAs from either WKY18-CH or SHRSP18-CH animals. Thus PAs responded to UCCAo with active vasodilation, but not structural remodeling, an effect that was absent in SHRSP. The increased tone of PAs from SHRSP animals, combined with lack of response to chronic hypoperfusion, may contribute to the propensity for ischemic lesions and increased perfusion deficit during hypertension.

Epidermal growth factor receptor is critical for angiotensin II-mediated hypertrophy in cerebral arterioles.

Angiotensin II (Ang II) is a major determinant of vascular remodeling in the cerebral circulation during chronic hypertension, which is an important risk factor for stroke. We examined the molecular mechanism of Ang II-mediated cerebrovascular remodeling that involves the epidermal growth factor receptor (EGFR) pathway. Mutant EGFR mice (waved-2), their heterozygous control (wild-type [WT]), and C57BL/6J mice were infused with Ang II (1000 ng kg(-1) min(-1)) or saline via osmotic minipumps for 28 days (n=8 per group). Eight of the Ang II-infused C57BL/6J mice were cotreated with AG1478 (12 mg/kg per day, IP), a specific EGFR tyrosine kinase inhibitor. Systolic arterial pressure was measured by a tail-cuff method. Pressure and diameter of cerebral arterioles were measured through an open cranial window in anesthetized mice. Cross-sectional area of the wall was determined in pressurized fixed cerebral arterioles. Expression of phosphorylated EGFR (p-EGFR), caveolin-1 (Cav-1), and c-Src was determined by western blotting and immunohistochemistry. Mutation of EGFR or AG1478 treatment did not affect Ang II-induced hypertension. Ang II increased the expression of p-EGFR in WT mice, confirming the activation of EGFR. Ang II induced hypertrophy and inward remodeling of cerebral arterioles in WT mice. Hypertrophy, but not remodeling, was prevented in waved-2 and AG1478-treated C57BL/6J mice. Ang II increased p-EGFR, Cav-1, and c-Src expression in WT but not in waved-2 or AG1478-treated C57BL/6J mice. These results suggest that Ang II-induced hypertrophy in cerebral arterioles involves EGFR-dependent signaling and may include Cav-1 and nonreceptor tyrosine kinase c-Src. This signaling pathway seems to be limited to Ang II-induced hypertrophy, but not inward remodeling, and is independent of blood pressure.

Magnesium sulfate treatment reverses seizure susceptibility and decreases neuroinflammation in a rat model of severe preeclampsia.

Eclampsia, defined as unexplained seizure in a woman with preeclampsia, is a life-threatening complication of pregnancy with unclear etiology. Magnesium sulfate (MgSO4) is the leading eclamptic seizure prophylactic, yet its mechanism of action remains unclear. Here, we hypothesized severe preeclampsia is a state of increased seizure susceptibility due to blood-brain barrier (BBB) disruption and neuroinflammation that lowers seizure threshold. Further, MgSO4 decreases seizure susceptibility by protecting the BBB and preventing neuroinflammation. To model severe preeclampsia, placental ischemia (reduced uteroplacental perfusion pressure; RUPP) was combined with a high cholesterol diet (HC) to cause maternal endothelial dysfunction. RUPP+HC rats developed symptoms associated with severe preeclampsia, including hypertension, oxidative stress, endothelial dysfunction and fetal and placental growth restriction. Seizure threshold was determined by quantifying the amount of pentylenetetrazole (PTZ; mg/kg) required to elicit seizure in RUPP + HC ± MgSO4 and compared to normal pregnant controls (n = 6/group; gestational day 20). RUPP+HC rats were more sensitive to PTZ with seizure threshold being ∼ 65% lower vs. control (12.4 ± 1.7 vs. 36.7 ± 3.9 mg/kg PTZ; p<0.05) that was reversed by MgSO4 (45.7 ± 8.7 mg/kg PTZ; p<0.05 vs. RUPP+HC). BBB permeability to sodium fluorescein, measured in-vivo (n = 5-7/group), was increased in RUPP+HC vs. control rats, with more tracer passing into the brain (15.9 ± 1.0 vs. 12.2 ± 0.3 counts/gram ×1000; p<0.05) and was unaffected by MgSO4 (15.6 ± 1.0 counts/gram ×1000; p<0.05 vs. controls). In addition, RUPP+HC rats were in a state of neuroinflammation, indicated by 35 ± 2% of microglia being active compared to 9 ± 2% in normal pregnancy (p<0.01; n = 3-8/group). MgSO4 treatment reversed neuroinflammation, reducing microglial activation to 6 ± 2% (p<0.01 vs. RUPP+HC). Overall, RUPP+HC rats were in a state of augmented seizure susceptibility potentially due to increased BBB permeability and neuroinflammation. MgSO4 treatment reversed this, increasing seizure threshold and decreasing neuroinflammation, without affecting BBB permeability. Thus, reducing neuroinflammation may be one mechanism by which MgSO4 prevents eclampsia during severe preeclampsia.

Postischemic reperfusion causes smooth muscle calcium sensitization and vasoconstriction of parenchymal arterioles.

BACKGROUND AND PURPOSE: Parenchymal arterioles (PAs) are high-resistance vessels in the brain that connect pial vessels to the microcirculation. We previously showed that PAs have increased vasoconstriction after ischemia and reperfusion that could increase perfusion deficit. Here, we investigated underlying mechanisms by which early postischemic reperfusion causes increased vasoconstriction of PAs. METHODS: Isolated and pressurized PAs from within the middle cerebral artery territory were studied in male Wistar rats that were either nonischemic control (n=34) or after exposure to transient middle cerebral artery occlusion (MCAO) by filament occlusion for 2 hours with 30 minutes of reperfusion (MCAO; n=38). The relationships among pressure-induced tone, smooth muscle calcium (using Fura 2), and membrane potential were determined. Sensitivity of the contractile apparatus to calcium was measured in permeabilized arterioles using Staphylococcus aureus α-toxin. Reactivity to inhibition of transient receptor potential melastanin receptor type 4 (9-phenanthrol), Rho kinase (Y27632), and protein kinase C (Gö6976) was also measured. RESULTS: After MCAO, PAs had increased myogenic tone compared with controls (47±2% versus 35±2% at 40 mm Hg; P<0.01), without an increase in smooth muscle calcium (177±21 versus 201±16 nmol/L; P>0.05) or membrane depolarization (-38±4 versus -36±1 mV; P>0.05). In α-toxin-permeabilized vessels, MCAO caused increased sensitivity of the contractile apparatus to calcium. MCAO did not affect dilation to transient receptor potential melastanin receptor type 4 or protein kinase C inhibition but diminished dilation to Rho kinase inhibition. CONCLUSIONS: The increased vasoconstriction of PAs during early postischemic reperfusion seems to be due to calcium sensitization of smooth muscle and could contribute to infarct expansion and limit neuroprotective agents from reaching their target tissue.

Increased pressure-induced tone in rat parenchymal arterioles vs. middle cerebral arteries: role of ion channels and calcium sensitivity.

Brain parenchymal arterioles (PAs) are high-resistance vessels that branch off pial arteries and perfuse the brain parenchyma. PAs are the target of cerebral small vessel disease and have been shown to have greater pressure-induced tone at lower pressures than pial arteries. We investigated mechanisms by which brain PAs have increased myogenic tone compared with middle cerebral arteries (MCAs), focusing on differences in vascular smooth muscle (VSM) calcium and ion channel function. The amount of myogenic tone and VSM calcium was measured using Fura 2 in isolated and pressurized PAs and MCAs. Increases in intraluminal pressure caused larger increases in tone and cytosolic calcium in PAs compared with MCAs. At 50 mmHg, myogenic tone was 37 ± 5% for PAs vs. 6.5 ± 4% for MCAs (P < 0.01), and VSM calcium was 200 ± 20 nmol/l in PAs vs. 104 ± 15 nmol/l in MCAs (P < 0.01). In vessels permeabilized with Staphylococcus aureus α-toxin, PAs were not more sensitive to calcium, suggesting calcium sensitization was not at the level of the contractile apparatus. PAs were 30-fold more sensitive to the voltage-dependent calcium channel (VDCC) inhibitor nifedipine than MCAs (EC50 for PAs was 3.5 ± 0.4 vs. 82.1 ± 2.1 nmol/l for MCAs;P < 0.01); however, electrophysiological properties of the VDCC were not different in VSM. PAs had little to no response to the calcium-activated potassium channel inhibitor iberiotoxin, whereas MCAs constricted ∼15%. Thus increased myogenic tone in PAs appears related to differences in ion channel activity that promotes VSM membrane depolarization but not to a direct sensitization of the contractile apparatus to calcium.