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Over the past decade there has been increasing support for a role of the immune system in the development of hypertension. Our lab has previously reported that female spontaneously hypertensive rats (SHRs) have a blood pressure (BP)-dependent increase in anti-inflammatory renal regulatory T cells (Tregs), corresponding to lower BP compared with males. However, little is known regarding the mechanism for greater renal Tregs in females. The current study was designed to test the hypothesis that the greater relative abundance of renal Tregs in female SHR is due to greater Treg production. To test this hypothesis, T cell profiles were measured in the spleen by flow cytometry in male and female SHR at 5 and 14 weeks of age. Splenic Tregs did not differ between males and females, suggesting sex differences in renal Tregs is not due to differences in production. To assess the role of the spleen in sex differences in renal Tregs and BP control, rats were randomized to receive sham surgery (CON) or splenectomy (SPLNX) at 12 weeks of age and implanted with telemeters to measure BP. After 2 weeks, kidneys were harvested for flow cytometric analysis of T cells. Splenectomy increased BP in both sexes after 2 weeks. Renal Tregs decreased in both sexes after splenectomy, abolishing the sex differences in renal Tregs. In conclusion, splenic Tregs were comparable in male and female SHRs, suggesting that sex differences in renal Tregs is due to differences in renal Treg recruitment, not Treg production.

Apoptosis is a physiological and anti-inflammatory form of cell death that is indispensable for normal physiology and homeostasis. Several studies have reported aberrant activation of apoptosis in various tissues at the onset of hypertension. However, the functional significance of apoptosis during essential hypertension remains largely undefined. The current study was designed to test the hypothesis that apoptosis contributes to sex differences in blood pressure and the T cell profile in spontaneously hypertensive rats (SHR). Apoptosis was measured in kidney, aorta and spleen of 13-week-old adult hypertensive male and female SHR. Female SHR had greater renal and aortic apoptosis compared to age-matched males; apoptosis in the spleen was comparable between the sexes. Based on well-established sex differences in hypertension, we tested the hypothesis that greater apoptosis in female SHR contributes to the lower BP and pro-inflammatory profile compared to males. Male and female SHR were randomized to receive vehicle or ZVAD-FMK, a cell permeable pan-caspase inhibitor, in established hypertension from 13 to 15 weeks of age or at the onset of hypertension from 6 to 12 weeks or age. Treatment with ZVAD-FMK lowered renal apoptosis in both studies, yet neither BP nor renal T cells were altered in either male or female SHR. These results suggest that apoptosis does not contribute to the control or maintenance of BP in male or female SHR or sex differences in renal T cells.

Hypertension remains the leading risk factor for cardiovascular disease. Young females tend to be protected from hypertension compared with age-matched males. Although it has become increasingly clear that the immune system plays a key role in the development of hypertension in both sexes, few studies have examined how cytokines mediate hypertension in males versus females. We previously published that there are sex differences in the levels of the cytokine tumor necrosis factor (TNF)-α in spontaneously hypertensive rats (SHR). The goal of this study was to test the hypothesis that TNF-α inhibition with etanercept will lower BP in male and female SHR. However, as male SHR have a more pro-inflammatory status than female SHR, we further hypothesize that males will have a greater decrease in BP with TNF-α inhibition than females. Young adult male and female SHR were administered increasing doses of the TNF-α inhibitor etanercept or vehicle twice weekly for 31 days and BP was continuously measured via telemetry. Following treatment, kidneys and urine were collected and analyzed for markers of inflammation and injury. Despite significantly decreasing renal TNF-α levels, renal phospho-NFκB and urinary MCP-1 excretion, etanercept did not alter BP in either male or female SHR. Interestingly, treatment with etanercept increased urinary excretion of protein, creatinine and KIM-1 in both sexes. These results indicate that TNF-α does not contribute to sex differences in BP in SHR but may be vital in the maintenance of renal health.

Background Compression therapy is the standard of care for venous ulcers and chronic venous insufficiency, but the treatment of underlying venous disease to relieve venous hypertension appears to improve wound healing and to decrease recurrence. Benefits of saphenous ablation have been well demonstrated; however, evidence for the treatment of incompetent perforating veins is less clear. Objective To assess the benefit of ultrasound-guided sclerotherapy (USGS) of incompetent perforator on healing of chronic venous ulcers. Patients and Methods This is a prospective cohort study which is a double arm clinical study to assess the healing rate for patients after USGFS vs standard compression therapy of patients with incompetent perforators veins within 6 month. Results Our preliminary data add further weight to the suggestion that eradication of SVR by means of UGFS improves CVU outcomes when compared to compression alone. In this regard, UGFS appears to be more effective more than compression therapy does, therefore, appear the more attractive option in this elderly patient population. Furthermore, the follow-up data presented here does suggest long-term healing following UGFS probably requires careful follow-up and, if required. Encouraged by these promising early results the authors have embarked upon a larger study to look at the role of UGFS in the treatment of CVU in greater depth. Conclusion Ultrasound guided foam sclerotherapy of perforator incompetent veins is a welltolerated and effective outpatient procedure. This study presents a patient cohort with limited exclusion criteria, allowing a representative cross section of VLU in general. Because of its higher healing rate and lower RR compared to other therapeutic options, and the improvement in quality of life that it brings, this minimally invasive, easily repeatable procedure may become the treatment of choice for VLU. Foam sclerotherapy is a simple procedure, compared to surgical intervention, no hospitalization, no anesthesia, can be done in outpatient clinic and the patient can return home after 45 min and no work off period. The relative freedom from serious complications and its evident success, makes this modality as a possible candidate for being the first line treatment for venous ulcers.

Numerous studies have demonstrated a sexual dimorphism in blood pressure (BP) control in spontaneously hypertensive rats (SHR), however the mechanisms remain to be further elucidated. Based on the established role of arachidonic acid metabolites and heme oxygenase (HO) in BP control, we hypothesize that higher BP in male SHR is associated with differential expression in renal HO and arachidonic acid metabolizing enzymes vs. female SHR. Higher BP in male SHR coincided with significant increases in renal cortical superoxide production and thiobarbituric acid reactive substances (TBARs) levels as measures of oxidative stress compared to normotensive female WKY and female SHR. The elevations in BP and oxidative stress in male SHR were also associated with a decrease in cortical heme oxygenase-1 (HO-1) expression when compared to normotensive female WKY. Although there was no sex or strain differences in cortical expression of the epoxyeicosatrienoic acids (EETs) producing enzyme, cytochrome P450 epoxygenase (CYP2C23), in male and female SHR and WKY, SHR had greater expression of the EETs metabolizing enzyme, soluble epoxide hydrolase (sEH) vs. WKY. Cortical expression of the 20-hydroxyeicosatetraenoic acid (20-HETE) producing enzyme, cytochrome P450 hydroxylase (CYP4A), was less in female WKY and SHR compared to strain-matched males and cortical 20-HETE levels were also less in female SHR vs. male SHR. Cortical cyclooxygenase-2 (COX-2) expression was significantly greater in female SHR and WKY vs. males and cortical prostaglandin E2 (PGE2) levels in female SHR was significantly greater than male WKY. In conclusion, our data suggest that sex differences in renal oxidative stress, HO-1 and arachidonic acid metabolizing enzymes could contribute to sexual dimorphism in hypertension in young SHR.

Diabetes is associated with increased risk and worsened outcome of stroke. Previous studies showed that male diabetic animals had greater hemorrhagic transformation (HT), profound loss of cerebral vasculature, and poor behavioral outcomes after ischemic stroke induced by suture or embolic middle cerebral artery occlusion (MCAO). Females are protected from stroke until reaching the menopause age, but young females with diabetes have a higher risk of stroke and women account for the majority of stroke mortality. The current study postulated that diabetes is associated with greater vascular injury and exacerbated sensorimotor and cognitive outcome after stroke even in young female animals. Male and female control and diabetic animals were subjected to transient MCAO and followed for 3 or 14 days to assess the neurovascular injury and repair. The vascularization indices after stroke were lower in male diabetic animals with 90-min but not 60-min ischemia/reperfusion injury, while there was no change in female groups. Cognitive deficits were exacerbated in both male and female groups regardless of the injury period, while the sensorimotor dysfunction was worsened in male diabetic animals with longer ischemia time. These results suggest that diabetes negates the protection afforded by sex in young female animals, and post-stroke vascularization pattern is influenced by the degree of injury and correlates with functional outcome in both sexes. Vasculoprotection after acute ischemic stroke may provide a novel therapeutic strategy in diabetes.

Core fucosylation (CF) of N-linked glycans is indispensable for normal physiology, but whether CF regulates any aspect of T cell activation, differentiation, or function remains unknown. Fucosyltransferase 8 (Fut8) is the only enzyme in mammals that catalyzes CF and following activation, both expression of Fut8 and CF synthesis increases on CD8+ T cells. To test the hypothesis that CF of N-glycans regulates memory T cell differentiation following acute viral infection, we generated mice with a floxed Fut8 gene. WT (CD4-Cre−) and Fut8−/− (CD4-Cre+) mice were infected with LCMV Armstrong (LCMV-Arm) and the differentiation of LCMV-specific CD8+ T cells was analyzed by measuring the surface expression of killer-cell lectin like receptor G1 (KLRG1) and the α chain of interleukin 7 receptor (CD127) to identify short-lived effector and memory precursor T cells, respectively. LCMV-specific CD8+ T cells from both WT and Fut8−/− followed the same pattern of expansion and contraction without any significant difference in the overall frequency of LCMV-specific CD8+ T cells. However, Fut8−/− retained a higher percentage of CD127+KLRG1− and a lower percentage of KLRG1+CD127− CD8 T cells throughout the course of infection suggesting CF influences the progression to long-term memory. At day 42 post infection, mice were infected with virulent Listeria monocytogenes expressing the LCMV epitope GP33–41. Both WT and Fut8−/− GP33-specific CD8+ T cells expanded equally. However, Fut8−/− retained a higher frequency of GP33-specific CD8+ T cells and did not undergo contraction until >3 weeks post-2ry challenge. Taken together, our findings suggest that CF of N-linked glycans regulates memory CD8+ T cell differentiation following acute viral infection. Supported by grants from NIH (R21 AI159401)

Post-stroke cognitive impairment (PSCI) is a major source of disability, affecting up to two thirds of stroke survivors with no available therapeutic options. The condition remains understudied in preclinical models due to its delayed presentation. Although hypertension is a leading risk factor for dementia, how ischemic stroke contributes to this neurodegenerative condition is unknown. In this study, we used a model of hypertension to study the development of PSCI and its mechanisms. Spontaneously hypertensive rats (SHR) were compared to normotensive rats and were subjected to 1-h middle cerebral artery occlusion or sham surgery. Novel object recognition, passive avoidance test and Morris water maze were used to assess cognition. In addition, brain magnetic resonance images were obtained 12-weeks post-stroke and tissue was collected for immunohistochemistry and protein quantification. Stroked animals developed impairment in long-term memory at 4-weeks post-stroke despite recovery from motor deficits, with hypertensive animals showing some symptoms of anhedonia. Stroked SHRs displayed grey matter atrophy and had a two-fold increase in apoptosis in the ischemic borderzone and increased markers of inflammatory cell death and DNA damage at 12 weeks post-stroke. This indicates that preexisting hypertension exacerbates the development of secondary neurodegeneration after stroke beyond its acute effects on neurovascular injury.

We previously published a sex difference in renal necrosis in SHR with males having a maturation induced increase in renal necrosis that is absent in females. Testosterone is known to drive an increase both in blood pressure (BP) with maturation in male SHR and necrosis of renal tubular epithelial cell in vitro. In the current study we tested the hypothesis that testosterone underlies the maturation induced increase in renal necrosis in male SHR. At 4 weeks of age, male SHR were randomly assigned to either sham or gonadectomy (ORX) groups (n=3). To control for the influence of high BP on renal necrosis, a third group was subjected to a sham surgery and treated with the anti-hypertensive medications hydrochlorothiazide (HCTZ; 55mg/kg/day) and reserpine (Res; 4.5 mg/kg/day) in drinking water starting at 9 weeks of age (n=4) to prevent age-related increases in BP. At 8 weeks of age, telemeters were implanted in all groups followed by a recovery for 1 week before BP was recorded. A separate set of rats were randomly assigned to Sham (n=3), ORX (n=4), or HCTZ/Res (n=4) and subjected to the exact same procedures as the previous set except for telemetry implantation. All rats were euthanized at 13 weeks of age and kidneys were collected for the quantification of renal necrosis using flow cytometric analysis of 7AAD + cells. Data were analyzed using one-way ANOVA and presented as mean ± standard error. BP was significantly lower in ODX and HCTZ/Res treated groups compared to sham (mean arterial blood pressure (mmHg): Sham= 139±2; HCTZ/Res= 117±3; ODX= 126±2; p=0.002; n=3-4). Renal necrosis was also significantly less in ORX rats, but not altered in HCTZ/Res treated groups compared to sham (renal necrosis expressed as % total gated kidney cells: Sham= 6±0.3%; HCTZ/Res=5±0.4%; ODX= 4±0.4%; P=0.003; n=6-7). Testosterone contributes to maturation induced increase in renal necrosis in male SHR.

Hypertension is the most common risk factor for cardiovascular disease, causing over 18 million deaths a year. Although the mechanisms controlling blood pressure (BP) in either sex remain largely unknown, T cells play a critical role in the development of hypertension. Further evidence supports a role for the immune system in contributing to sex differences in hypertension. The goal of the current study was to first, determine the impact of sex on the renal T-cell profiles in DOCA-salt hypertensive males and females and second, test the hypothesis that greater numbers of T regulatory cells (Tregs) in females protect against DOCA-salt–induced increases in BP and kidney injury. Male rats displayed greater increases in BP than females following 3 weeks of DOCA-salt treatment, although increases in renal injury were comparable between the sexes. DOCA-salt treatment resulted in an increase in proinflammatory T cells in both sexes; however, females had more anti-inflammatory Tregs than males. Additional male and female DOCA-salt rats were treated with anti-CD25 to decrease Tregs. Decreasing Tregs significantly increased BP only in females, thereby abolishing the sex difference in the BP response to DOCA-salt. This data supports the hypothesis that Tregs protect against the development of hypertension and are particularly important for the control of BP in females.

Necrosis is a pathological form of cell death that induces an inflammatory response, and immune cell activation contributes to the development and maintenance of hypertension. Necrosis was measured in kidney, spleen, and aorta of 12- to 13-week-old male and female SHRs (spontaneously hypertensive rats); male SHRs had greater renal necrotic cell death than female SHRs. Because male SHRs have a higher blood pressure (BP) and a more proinflammatory T-cell profile than female SHRs, the current studies tested the hypothesis that greater necrotic cell death in male SHRs exacerbates increases in BP and contributes to the proinflammatory T-cell profile. Male and female SHRs were randomized to receive vehicle or Necrox-5—a cell permeable inhibitor of necrosis—from 6 to 12 weeks of age or from 11 to 13 weeks of age. In both studies, Necrox-5 decreased renal necrosis and abolished the sex difference. Treatment with Necrox-5 beginning at 6 weeks of age attenuated maturation-induced increases in BP in male SHR; BP in female SHR was not altered by Necrox-5 treatment. Necrox-5 decreased proinflammatory renal T cells in both sexes, although sex differences were maintained. Administration of Necrox-5 for 2 weeks in SHR with established hypertension resulted in a small but significant decrease in BP in males with no effect in females. These results suggest that greater necrotic cell death in male SHR exacerbates maturation-induced increases in BP with age contributing to sex differences in BP. Moreover, although necrosis is proinflammatory, it is unlikely to explain sex differences in the renal T-cell profile.

It is a clinically well-established fact that patients with diabetes have very poor stroke outcomes. Yet, the underlying mechanisms are largely unknown. Previously we have shown that male diabetic animals show greater hemorrhagic transformation (HT), profound loss of cerebral vasculature in the recovery period and poor outcomes after ischemic stroke induced by a 90-min or embolic middle cerebral artery occlusion (MCAO). Given that young females have a 3.5-fold higher risk of stroke with the onset of diabetes and women account for the majority of stroke mortality, the current study postulated that diabetes causes greater vascular injury/repair and worsens sensorimotor and cognitive recovery after stroke even in young females. After 8 weeks of type 2 diabetes induced by a combination of high fat diet and low dose streptozotocin (30mg/kg;IP), Wistar rats were subjected to 60-min MCAO and followed for 3 or 14 days for evaluation of neurovascular injury (infarct size, HT, edema) and repair (vascularization patterns and functional outcomes), respectively. Vascular indices measured included branch density, tortuosity, vascular volume and surface area in cortex and striatum using confocal imaging of FITC-filled vasculature. Males had comparable infarct size and edema regardless of disease while diabetic females had greater infarction/edema compared to control. In contrast to our previous studies, there was no difference in HT or vascularization patterns between control and diabetic animals. All groups recovered from functional deficits to the same degree. These results 1) provide evidence that diabetes negates neuroprotection typically seen in female animals, 2) strongly suggest that HT impacts recovery responses and 3) post-stroke vascularization pattern is influenced by the degree of acute vascular injury and correlates with functional recovery in both sexes. Treatment of bleeding that occurs as a result of HT may provide a novel therapeutic strategy in diabetes.