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Chronic interstitial inflammation and renal infiltration of activated immune cells play an integral role in hypertension. Lymphatics regulate inflammation through clearance of immune cells and excess interstitial fluid. Previously, we demonstrated increasing renal lymphangiogenesis prevents hypertension in mice. We hypothesized that targeted nanoparticle delivery of vascular endothelial growth factor-C (VEGF-C) to the kidney would induce renal lymphangiogenesis, lowering blood pressure in hypertensive mice. A kidney-targeting nanoparticle was loaded with a VEGF receptor-3-specific form of VEGF-C and injected into mice with angiotensin II-induced hypertension or LNAME-induced hypertension every 3 days. Nanoparticle-treated mice exhibited increased renal lymphatic vessel density and width compared to hypertensive mice injected with VEGF-C alone. Nanoparticle-treated mice exhibited decreased systolic blood pressure, decreased pro-inflammatory renal immune cells, and increased urinary fractional excretion of sodium. Our findings demonstrate that pharmacologically expanding renal lymphatics decreases blood pressure and is associated with favorable alterations in renal immune cells and increased sodium excretion.

Salt-induced hypertension is associated with chronic interstitial inflammation and increases in activated inflammatory immune cells in the kidney. It is currently unknown if these renal immune cell changes result from high blood pressure or directly from high salt concentrations. Using a salt-sensitive hypertension (SSH) mouse model and hydralazine (HDZ), we hypothesized that some renal immune cells are affected by high blood pressure, and some will be affected by the high salt content without increased systemic pressure. Three groups of mice (n=5 in each) were generated and given L-NAME via drinking water for 2 weeks, with two groups receiving a high salt (4%) diet for 3 weeks after a 2-week washout, and one of these receiving HDZ (250 mg/L) via drinking water simultaneously during the high salt diet. Systolic blood pressure was significantly decreased in the SSH group treated with HDZ, dropping to 119±1 mmHg from 141±1 mmHg (p

We recently reported that increased proinflammatory (M1) macrophages, inflammation, and inflammation-associated lymphangiogenesis is associated with reproductive dysfunction in mice with salt-sensitive HTN (SSHTN). However, it is unknown whether these effects are primarily due to increased systemic pressure and/or salt. We hypothesized that artificial lowering of blood pressure may not reverse the inflammatory environment within the testes, that is associated with SSHTN. Male C57BL6/J mice were made hypertensive by providing L-NAME (0.5 mg/mL) in the drinking water for 2 weeks, followed by a 2-week washout period, and a subsequent 3-week 4% high salt diet (SSHTN). In another group, mice received L-NAME 2 weeks, followed by a 2-week washout period and a subsequent 3-week 4% high salt diet along with hydralazine (250mg/L) in their drinking water (SSHTN+HDZ). Control (C) mice received L-NAME for 2 weeks and then tap water and normal diet. Systolic blood pressure in SSHTN mice (141±1 mm Hg) was significantly increased when compared to C mice (102±3 mm Hg), but the pressure decreased to 119±1 mm Hg (p

Hypertension affects roughly half of the U.S. population and is characterized by detrimental pro-inflammatory changes that alter renal function and the cardiovascular system over time. The kidney is a major regulator of blood pressure, however, the specific contributions of renal cell types, including lymphatic endothelial cells (LECs), to hypertension have not been thoroughly explored. Previous work from our lab has shown an increase in renal lymphangiogenesis in response to hypertension and that augmentation of renal lymphangiogenesis can reduce blood pressure. CCN1/Cyr61 is an extracellular matrix protein secreted by multiple cell types, particularly LECs and fibroblasts, which contributes to integrin signaling pathways. CCN1 is also known to be involved in lymphangiogenesis, and has previously been implicated in GWAS studies of hypertension. Given these connections, we performed single cell RNA sequencing using angiotensin II and salt sensitive mouse models of hypertension, which were analyzed separately as well as aggregated into one hypertensive group. Kidneys were turned to a single cell suspension and separated for CD31+/Podoplanin+ cells meant to better represent LECs, which are a small fraction of renal cells. Sequencing was performed and the gene reads were run through the Cell Ranger, Seurat, FGSEA, and Slingshot pipelines, then CCN1+ cells were isolated digitally and analyzed separately. In the AngII model, a total of 60 genes (28 up, 32 down) were differentially expressed (p3 or

Background: Hypertension (HTN) is associated with renal proinflammatory immune cell infiltration and increased sodium retention. We reported previously that renal lymphatic vessels, which are responsible for trafficking immune cells from the interstitial space to draining lymph nodes, increase in density under hypertensive conditions. We also demonstrated that augmenting renal lymphatic density can prevent HTN in mice. Whether renal lymphangiogenesis can treat HTN in mice is unknown. We hypothesized that genetically inducing renal lymphangiogenesis after the establishment of HTN would attenuate HTN in male and female mice from three different HTN models. Methods: Mice with inducible kidney-specific overexpression of VEGF-D (KidVD) experience renal lymphangiogenesis upon doxycycline administration. HTN was induced in KidVD+ and KidVD- mice by subcutaneous release of angiotensin II, administration of the nitric oxide synthase inhibitor L-NAME, or consumption of a 4% salt diet following a L-NAME priming and washout period. After a week of HTN stimuli treatment, doxycycline was introduced. Systolic blood pressure (SBP) readings were taken weekly. Kidney function was determined from urine and serum measures. Kidneys were processed for RT-qPCR, flow cytometry, and imaging. Results: Mice that underwent renal-specific lymphangiogenesis had significantly decreased SBP and renal proinflammatory immune cells. Additionally, renal lymphangiogenesis was associated with a decrease in sodium transporter expression and increased fractional excretion of sodium, indicating improved sodium handling efficiency. Conclusions: These findings demonstrate that augmenting renal lymphangiogenesis can treat HTN in male and female mice by improving renal immune cell trafficking and sodium handling.

The lymphatic vessels play an essential role in maintaining immune and fluid homeostasis, and in the transport of dietary lipids. The discovery of lymphatic endothelial cell-specific markers facilitated the visualization and mechanistic analysis of lymphatic vessels over the past two decades. As a result, lymphatic vessels have emerged as a crucial player in the pathogenesis of several cardiovascular diseases, as demonstrated by worsened disease progression caused by perturbations to lymphatic function. In this review, we discuss the major findings on the role of lymphatic vessels in cardiovascular diseases such as hypertension, obesity, atherosclerosis, myocardial infarction, and heart failure.

Renal innate immune cell accumulation and inflammation are associated with hypertension. Time restricted feeding (TRF) has been reported to decrease inflammation and blood pressure. Whether TRF can decrease blood pressure by decreasing renal innate immune cells in hypertension is unknown.We determined whether TRF can decrease blood pressure in two separate mouse models of hypertension, N(G)-nitro-L-arginine methyl ester hydrochloride-induced hypertension (LHTN) and salt-sensitive hypertension (SSHTN). Once hypertension was established after 2 days, TRF (12-h food/12-h no food) for 4 weeks significantly decreased systolic blood pressure in both LHTN and SSHTN mice despite no differences in the amount of food eaten or body weight between groups. Activated macrophages and dendritic cells in the kidneys of both LHTN and SSHTN mice were decreased significantly in mice that underwent TRF. This was associated with an improvement in kidney function (decreased serum creatinine, decreased fractional excretion of sodium, and increased creatinine clearance) which achieved significance in LHTN mice and trended towards improvement in SSHTN mice.Our findings demonstrate that TRF can significantly decrease renal innate immune cells and blood pressure in two mouse models of hypertension.

The contribution of immune cells in the initiation and maintenance of hypertension is undeniable. Several studies have established the association between hypertension, inflammation, and immune cells from the innate and adaptive immune systems. Here, we provide an update to our 2017 American Journal of Hypertension review on the overview of the cellular immune responses involved in hypertension. Further, we discuss the activation of immune cells and their contribution to the pathogenesis of hypertension in different in vivo models. We also highlight existing gaps in the field of hypertension that need attention. The main goal of this review is to provide a knowledge base for translational research to develop therapeutic strategies that can improve cardiovascular health in humans.

Hypertension (HTN) is associated with gonadal dysfunction and impaired reproductive health in both men and women. An imbalance in the systemic and renal proinflammatory (M1)/anti-inflammatory (M2) macrophage ratio, increased inflammation, and inflammation-associated lymphangiogenesis have been observed in animals with HTN. However, the impact of HTN on gonadal macrophages, inflammation, and lymphatics remains obscure. We hypothesized that salt-sensitive HTN (SSHTN) and HTN alters gonadal macrophage polarization, which is associated with inflammation, inflammation-associated lymphangiogenesis, and reproductive dysfunction. Flow cytometry analyses revealed a significant increase in M1 macrophages in the testes of SSHTN and nitro-L-arginine methyl ester hydrochloride (L-NAME)-induced HTN (LHTN) mice, with a concurrent decrease in M2 macrophages in SSHTN mice yet an increase in M2 macrophages in LHTN mice. Ovaries from SSHTN mice exhibited an increase in M1 and a decrease in M2 macrophages, while ovaries from LHTN mice had a significant increase in M2 and a decrease in M1 macrophages. Gene expression patterns of proinflammatory cytokines revealed gonadal inflammation in all hypertensive mice. Increased lymphatic vessel density in the gonads of both male and female hypertensive mice was confirmed by immunofluorescence staining for lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1). HTN adversely affected the expression pattern of steroidogenic enzymes, hormone receptors, and secretory proteins in both the testes and ovaries. In line with these results, male hypertensive mice also presented with decreased sperm concentration, and increased percentage of sperm with abnormal morphology, damaged acrosome, and nonfunctional mitochondrial activity. These data demonstrate that HTN alters gonadal macrophage polarization, which is associated with gonadal inflammation, inflammation-associated lymphangiogenesis, and dysfunction.

OBJECTIVE: Hypertension is associated with renal immune cell accumulation and sodium retention. Lymphatic vessels provide a route for immune cell trafficking and fluid clearance. Whether specifically increasing renal lymphatic density can treat established hypertension, and whether renal lymphatics are involved in mechanisms of blood pressure regulation remain undetermined. Here, we tested the hypothesis that augmenting renal lymphatic density can attenuate blood pressure in established hypertension. METHODS: Transgenic mice with inducible kidney-specific overexpression of VEGF-D (‘KidVD+’ mice) and KidVD–controls were administered a nitric oxide synthase inhibitor, l-NAME, for 4 weeks, with doxycycline administration beginning at the end of week 1. To identify mechanisms by which renal lymphatics alter renal Na(+) handling, Na(+) excretion was examined in KidVDR mice during acute and chronic salt loading conditions. RESULTS: Renal VEGF-D induction for 3 weeks enhanced lymphatic density and significantly attenuated blood pressure in KidVDR mice whereas KidVD mice remained hypertensive. No differences were identified in renal immune cells, however, the urinary Na(+) excretion was increased significantly in KidVD+ mice. KidVD+ mice demonstrated normal basal sodium handling, but following chronic high salt loading, KidVD+ mice had a significantly lower blood pressure along with increased urinary fractional excretion of Na(+). Mechanistically, KidVD+ mice demonstrated decreased renal abundance of total NCC and cleaved ENaCα Na(+) transporters, increased renal tissue fluid volume, and increased plasma ANP. CONCLUSION: Our findings demonstrate that therapeutically augmenting renal lymphatics increases natriuresis and reduces blood pressure under sodium retention conditions.

Macrophages are the principal immune cells in ovaries. Besides protecting against invading pathogens and antigens, macrophages also play an essential role in folliculogenesis, ovulation, luteinization, lymphangiogenesis, and other functions. An imbalance in M1/M2 macrophages is observed in systemic circulation in patients and animals with hypertension (HTN). Although studies have demonstrated an association between HTN and reproductive dysfunction in females, the effect of HTN on ovarian M1/M2 ratio and lymphatics is largely unknown. We hypothesized that L-NAME-induced HTN (LHTN) and salt-sensitive hypertension (SSHTN) may increase the M1/M2 balance in ovaries which is associated with lymphangiogenesis and reproductive dysfunction in mice. Female mice were either provided L-NAME (0.5 mg/mL) in their drinking water for 3 weeks or L-NAME for 2 weeks, followed by a 2-week washout period and subsequent 3-week 4% high salt diet (SSHTN). Control mice (C) received tap water and normal diet. Flow cytometry analysis revealed a significant decrease in M1 (C: 46%±1, LHTN: 33%±2; p

Elevated circulating proinflammatory (M1) and decreased anti-inflammatory (M2) macrophages contribute to hypertension (HTN) and end-organ damage. HTN is associated with reproductive dysfunction in men. However, the impact of HTN on testicular macrophages and inflammation is unknown. We hypothesized that HTN increases M1 and decreases M2 testicular macrophages, which is associated with inflammation and reproductive dysfunction. Male mice were made hypertensive by either providing L-arginine methyl ester hydrochloride (L-NAME) (0.5 mg/mL) in the drinking water for 3 weeks [L-NAME-induced HTN (LHTN)] or L-NAME water for 2 weeks, followed by a 2-week washout period and a subsequent 3-week 4% high salt diet [salt-sensitive hypertension (SSHTN)]. Control (C) mice received tap water and normal diet. Flow cytometry analysis revealed a significant increase in both M1 (C: 15%±1, LHTN: 22%±2; p

Recent studies suggest that the microbiome plays a key role in hypertension and associated inflammation. Microbiota produce metabolites that may lead to activated pro-inflammatory immune cells and contribute to hypertension; however, the altered metabolites in multiple models of hypertension is currently unknown. We hypothesized that there are significant differences in metabolomic profiles between normotensive and hypertensive mice. We utilized two mouse models of hypertension: L-arginine methyl ester hydrochloride (L-NAME)/high salt diet induced hypertension (LSHTN) and angiotensin II induced hypertension (A2HTN). Serum and fecal samples were collected at the end of the treatment period. Ultra-high performance liquid chromatography and tandem mass spectrometry were performed to identify the biochemical composition of each sample. Random Forest Analysis was performed to classify each sample based on similarities and differences in metabolite composition. These procedures were performed by Metabolon, Inc. A total of 1,066 and 1,028 biochemicals were measured in serum and feces, respectively. There were 263 biochemicals in LSHTN serum and 122 biochemicals in A2HTN serum that were statistically different from controls (p≤0.05). There were 298 biochemicals in LSHTN feces and 64 biochemicals in A2HTN feces that were statistically different from controls (p≤0.05). Five biochemical metabolite groups were shown to have significant differences between hypertensive groups and controls: aromatic amino acids, bile acids and sterols, benzoates, fatty acids, and diacylglycerols. Tryptophan metabolites were significantly reduced in the serum of LSHTN mice but not in the serum of A2HTN mice. Serum tyrosine and benzoate metabolites showed varied differences between the two hypertensive groups. Serum fatty acid beta oxidation metabolites were significantly reduced in both hypertensive models but were significantly increased in the feces of mice with LSHTN. In conclusion, this study provided significant analysis of metabolite changes in two hypertension mouse models. Further investigation of the roles these metabolites play in hypertension may lead to targeted therapeutic interventions.

Metabolomic Study to Identify Common Metabolites in Two Different Mouse Models of Hypertension Recent studies have reflected the importance of the body’s microbiome and associated metabolites and their changes in hypertension. In the current study, we hypothesized that metabolite changes common between hypertension models may unify hypertension’s pathophysiology with respect to metabolites. Two different mice models of experimental hypertension were used in the study: (1) L-arginine methyl ester hydrochloride (L-NAME)/High salt diet induced hypertension (LSHTN) and (2) angiotensin II induced hypertension (AHTN). Untargeted global metabolomics analysis in serum and urine samples were performed to identify common metabolites altered across both hypertensive models, and the resulting data were analyzed using MetaboAnalyst software and compared to control mice. A list of metabolites that were altered significantly in both models of hypertension were identified. A total of 41 serum metabolites were identified as being altered significantly in any hypertensive model compared to controls. Of these, however, only 4 were increased significantly, and 10 were decreased significantly in common across both hypertensive groups. In the urine, 6 metabolites were altered significantly in any hypertensive group with respect to control, however, 0 of them were common between the hypertensive groups. These findings demonstrate that a modest, but potentially important, number of serum metabolites are commonly altered between experimental hypertension models. Further studies to understand the role of these identified metabolites may lead to a greater understanding of the association between gut dysbiosis and hypertension. Submitted to American Heart Association Council on Hypertension Scientific Sessions (September 27-29, 2021, Virtual)Abstract#: 21-HBPR-A-578-AHA

Hypertension is associated with inflammation and decreased kidney function. Studies of cardiovascular function have observed that time-restricted feeding (TRF), a form of intermittent fasting, is associated with decreased blood pressure, decreased inflammation, and improved kidney function. We hypothesized that implementation of a time-restricted feeding protocol in hypertensive mice would decrease systolic blood pressure and increase kidney function. C57BL6/J mice were randomly assigned to either an L-arginine methyl ester hydrochloride (LNAME)-induced hypertension (LHTN) model, where they received LNAME in their drinking water, or a salt-sensitive hypertension (SSHTN) model, where they received a 4% high salt diet following LNAME priming and a washout period. Two days following introduction of LNAME or the high salt diet, mice were either provided food ad libitum or placed on a 12-hour TRF protocol, where they were only allowed to eat from 8PM to 8AM. Hypertensive mice receiving TRF treatment displayed a significantly decreased systolic blood pressure (SBP) after 4 weeks when compared to the control hypertensive groups (LHTN SBP: 164±1 vs. 149±2 mmHg, p

BACKGROUND Renal inflammation and immune cell infiltration are characteristic of several forms of hypertension. Our laboratory has previously demonstrated that renal-inflammation-associated lymphangiogenesis occurs in salt-sensitive and nitric-oxide-inhibition-induced hypertension. Moreover, enhancing renal lymphatic density prevented the development of these two forms of hypertension. Here, we investigated the effects of angiotensin II-induced hypertension on renal lymphatic vessel density in male and female mice. METHODS Wild-type and genetically engineered male and female mice were infused with angiotensin II for 2 or 3 weeks. Isolated splenocytes and peritoneal macrophages from mice, and commercially available mouse lymphatic endothelial cells were used for in vitro studies. RESULTS Compared to vehicle controls, angiotensin II-infused male and female mice had significantly increased renal lymphatic vessel density in association with pro-inflammatory immune cells in the kidneys of these mice. Direct treatment of lymphatic endothelial cells with angiotensin II had no effect as they lack angiotensin II receptors; however, angiotensin II treatment of splenocytes and peritoneal macrophages induced secretion of the lymphangiogenic growth factor VEGF-C in vitro. Utilizing our genetic mouse model of inducible renal lymphangiogenesis, we demonstrated that greatly augmenting renal lymphatic density prior to angiotensin II infusion prevented the development of hypertension in male and female mice and this was associated with a reduction in renal CD11c+F4/80- monocytes. CONCLUSION Renal lymphatics play a significant role in renal immune cell trafficking and blood pressure regulation, and represent a novel avenue of therapy for hypertension.

Hypertension is one of the most prevalent diseases that leads to end organ damage especially affecting the heart, kidney, brain, and eyes. Numerous studies have evaluated the association between hypertension and impaired sexual health, in both men and women. The detrimental effects of hypertension in men includes erectile dysfunction, decrease in semen volume, sperm count and motility, and abnormal sperm morphology. Similarly, hypertensive females exhibit decreased vaginal lubrication, reduced orgasm, and several complications in pregnancy leading to fetal and maternal morbidity and mortality. The adverse effect of hypertension on male and female fertility is attributed to hormonal imbalance and changes in the gonadal vasculature. However, mechanistic studies investigating the impact of hypertension on gonads in more detail on a molecular basis remain scarce. Hence, the aim of the current review is to address and summarize the effects of hypertension on reproductive health, and highlight the importance of research on the effects of hypertension on gonadal inflammation and lymphatics.

Hypertension (HTN) is associated with reduced fertility in men. Although numerous studies report that HTN disrupts hormonal balance in men, less is known about the direct effect of HTN on testes and how HTN influences testicular inflammation and lymphatics. We hypothesized that HTN increases testicular lymphatic vessel density and this is associated with immune cell infiltration and inflammation. Male mice (8 weeks old) were made hypertensive by providing them with L-arginine methyl ester hydrochloride (L-NAME) (0.5 mg/mL) in the drinking water for 3 weeks and control mice received tap water. Testes of hypertensive mice had a significant increase in gene expression of the lymphatic vessel markers Lyve-1 (17.7 ± 2.1 fold; p

Chronic interstitial inflammation and renal infiltration of activated immune cells play an integral role in hypertension. Lymphatic vessels attenuate inflammation by trafficking activated immune cells and excess fluid from the interstitial space to lymph nodes. Previously, our laboratory demonstrated that genetically inducing renal lymphangiogenesis could treat hypertension in three different mouse models. In an effort to translate these findings into a clinical treatment, we hypothesized that a targeted nanoparticle could deliver the pro-lymphangiogenic factor VEGF-C156S to the kidney, induce lymphangiogenesis, and lower blood pressure in hypertensive mice. A micellar nanoparticle was developed with the capacity to deliver protein to the kidney, as demonstrated through delivery trials. This nanoparticle was loaded with VEGF-C156S and injected into mice with LNAME-induced hypertension (LHTN) or angiotensin II-induced hypertension (AIIHTN) via tail vein every 3 days. Compared to hypertensive mice injected with VEGF-C156S only (no nanoparticle) every 3 days, nanoparticle-treated mice exhibited a significantly lower systolic blood pressure (SBP) after 4 injections (LHTN SBP: 160±5 vs. 120±3 mmHg, p

Objective Innate immune system activation and excessive inflammation contributes to hypertension during pregnancy (HTN-preg). Activation of Toll-like receptors (TLRs), the primary innate immune system sensor, is evident in women with HTN-preg and is sufficient to induce pregnancy-dependent, proteinuric hypertension in animals. However, whether HTN-preg is a maternal disease, a placental disease, or both is unclear. We hypothesized that activation of TLR3, the double-stranded RNA sensor, in both maternal systemic and placental cells would be necessary for the full development of HTN-preg in mice. Study design Various mating schemes generated pregnant mice that lacked TLR3 in maternal cells, paternally-derived placental cells, and both. Mice were then injected with a TLR3 agonist on days 13, 15, and 17 of pregnancy. Main outcome measures Blood pressure, urinary protein excretion, fetal development, maternal vascular endothelial function, and immune system activation were all assessed and compared between groups. Results Pregnant mice lacking TLR3 in maternal cells as well as pregnant mice lacking TLR3 in placental cells had significantly attenuated increases in systolic blood pressure, urinary protein excretion, fetal demise, and endothelial dysfunction compared to wild-type pregnant mice following TLR3 activation. Pregnant mice lacking TLR3 in both maternal systemic and placental cells were completely resistant to the hypertension, proteinuria, fetal demise, endothelial dysfunction, splenomegaly, and increases in pro-inflammatory immune cells induced by TLR3 activation. Conclusions These data suggest that both maternal and placental TLR3 activation are crucial for the full development of HTN-preg and that TLR3 antagonists may be beneficial in some women with HTN-preg.

The calcineurin inhibitor cyclosporine A suppresses the immune system but promotes hypertension, vascular dysfunction, and renal damage. Cyclosporine A decreases regulatory T cells and this contributes to the development of hypertension. However, cyclosporine A’s effects on another important regulatory immune cell subset, myeloid-derived suppressor cells (MDSCs), is unknown. We hypothesized that augmenting MDSCs would ameliorate the cyclosporine A-induced hypertension and vascular and renal injury and dysfunction and that cyclosporine A reduces MDSCs in mice. Daily interleukin-33 treatment, which increased MDSC levels, completely prevented cyclosporine A-induced hypertension and vascular and renal toxicity. Adoptive transfer of MDSCs from control mice into cyclosporine A-treated mice after hypertension was established dose-dependently reduced blood pressure and vascular and glomerular injury. Cyclosporine A treatment of aortas and kidneys isolated from control mice for 24 hours decreased relaxation responses and increased inflammation, respectively, and these effects were prevented by the presence of MDSCs. MDSCs also prevented the cyclosporine A-induced increase in fibronectin in microvascular and glomerular endothelial cells. Lastly, cyclosporine A dose-dependently reduced the number of MDSCs by inhibiting calcineurin and preventing cell proliferation, as other direct calcineurin signaling pathway inhibitors had the same dose-dependent effect. These data suggest that augmenting MDSCs can reduce the cardiovascular and renal toxicity and hypertension caused by cyclosporine A.

Aberrant innate immune system activation in the mother contributes greatly to the development of hypertension during pregnancy. Numerous groups have elicited vascular inflammation, endothelial dysfunction, and hypertension in animals during gestation by directly activating Toll-like receptors. Additionally, several experimental therapies that reduce pro-inflammatory immune cells and cytokines restore vascular endothelial function and normalize blood pressure. This review will summarize the research demonstrating that an excessive maternal innate immune response is sufficient to cause vascular inflammation and endothelial dysfunction, which contributes to the development of hypertension during pregnancy. Dampening the vascular inflammation caused by immune responses may reduce the incidence and severity of hypertensive disorders of pregnancy.

The immunosuppressive calcineurin inhibitors cyclosporine A and tacrolimus alter T-cell subsets and can cause hypertension, vascular dysfunction, and renal toxicity. We and others have reported that cyclosporine A and tacrolimus decrease anti-inflammatory regulatory T cells and increase proinflammatory interleukin-17–producing T cells; therefore, we hypothesized that inhibition of these effects using noncellular therapies would prevent the hypertension, endothelial dysfunction, and renal glomerular injury induced by calcineurin inhibitor therapy. Daily treatment of mice with cyclosporine A or tacrolimus for 1 week significantly decreased CD4 + /FoxP3 + regulatory T cells in the spleen and lymph nodes, as well as induced hypertension, vascular injury and dysfunction, and glomerular mesangial expansion in mice. Daily cotreatment with all-trans retinoic acid reported to increase regulatory T cells and decrease interleukin-17–producing T cells, prevented all of the detrimental effects of cyclosporine A and tacrolimus. All-trans retinoic acid also increased regulatory T cells and prevented the hypertension, endothelial dysfunction, and glomerular injury in genetically modified mice that phenocopy calcineurin inhibitor–treated mice (FKBP12-Tie2 knockout). Treatment with an interleukin-17–neutralizing antibody also increased regulatory T-cell levels and prevented the hypertension, endothelial dysfunction, and glomerular injury in cyclosporine A–treated and tacrolimus-treated mice and FKBP12-Tie2 knockout mice, whereas an isotype control had no effect. Augmenting regulatory T cells and inhibiting interleukin-17 signaling using noncellular therapies prevents the cardiovascular and renal toxicity of calcineurin inhibitors in mice.

Sodium retention is a hallmark of most forms of experimental hypertension. Renal lymphatics maintain fluid homeostasis by draining cortical interstitial fluid to the draining lymph node. Our lab has previously demonstrated that augmenting renal lymphatics prevents hypertension, but whether this can treat hypertension and whether expanding renal lymphatics alters renal sodium handling remain unknown. Our hypotheses were that augmenting renal lymphatic vessel density after hypertension is established will treat salt sensitive hypertension (SSHTN) and angiotensin II-induced hypertension (AIIHTN) and that this will be accompanied by an increase in urinary sodium excretion. To test our hypotheses, we utilized transgenic mice that overexpress the lymphangiogenic growth factor VEGF-D specifically in the kidney when administered doxycycline (KidVD+ mice). KidVD+ mice and KidVD- littermates were made salt-sensitive by treatment with L-NAME (0.5 mg/mL) for two weeks, followed by a washout period of two weeks, and then given a 4% high salt diet for four weeks. To induce AIIHTN, mice were infused with angiotensin II (490 ng/kg/min) for four weeks. Doxycycline was administered to all mice a week after beginning the high salt diet or a week after angiotensin II infusion. Prior to doxycycline initiation, KidVD- and KidVD+ mice were hypertensive (SSHTN SBP: 130±2 and 134±2 mmHg, respectively; AIIHTN SBP: 125±2 and 128±2 mmHg, respectively). Compared to KidVD- mice, doxycycline administration for three weeks augmented renal lymphatics in KidVD+ mice and significantly decreased blood pressure after four weeks of high salt diet or angiotensin II (SSHTN SBP: 134±4 vs. 125±2 mmHg; p

Renal immune cell infiltration and accompanying inflammation activates renal sodium transporters leading to sodium retention and hypertension. Lymphatic vessels traffic interstitial immune cells to the draining lymph nodes and help resolve inflammation. We have previously demonstrated that genetically inducing renal lymphangiogenesis prevents hypertension associated with a reduction in renal immune cell accumulation; however, it is unknown whether augmenting renal lymphatics can treat hypertension. Our hypothesis was that augmenting renal lymphatics after hypertension is established will lower blood pressure during L-NAME-induced hypertension (LHTN). Transgenic mice that overexpress the lymphangiogenic signal VEGF-D only in the kidney upon doxycycline administration (KidVD+ mice) and KidVD- littermates were administered L-NAME (0.5 mg/mL) in their drinking water for four weeks with doxycycline initiated at the second week of L-NAME. Treatment with L-NAME for one week induced LHTN in both KidVD- and KidVD+ mice (SBP: 134±4 and 142±5 mmHg, respectively). However, doxycycline-induced renal lymphangiogenesis significantly decreased blood pressure in KidVD+ mice compared to KidVD- mice after four weeks of L-NAME (SBP: 127±5 vs. 151±6 mmHg; p+ F4/80 - monocytes and a significant increase in 24-hour urinary sodium excretion and fractional excretion of sodium in KidVD+ mice. Urinary volume over 24 hours was also increased in KidVD+ mice (1.7±0.8 vs. 3±0.9 mL/24 hours; p

Pre-eclampsia, the development of hypertension and proteinuria or end-organ damage during pregnancy, is a leading cause of both maternal and fetal morbidity and mortality, and there are no effective clinical treatments for pre-eclampsia aside from delivery. The development of pre-eclampsia is characterized by maladaptation of the maternal immune system, excessive inflammation and endothelial dysfunction. We have reported that detection of extracellular RNA by the Toll-like receptors (TLRs) 3 and 7 is a key initiating signal that contributes to the development of pre-eclampsia. PLacental eXpanded (PLX-PAD) cells are human placenta-derived, mesenchymal-like, adherent stromal cells that have anti-inflammatory, proangiogenic, cytoprotective and regenerative properties, secondary to paracrine secretion of various molecules in response to environmental stimulation. We hypothesized that PLX-PAD cells would reduce the associated inflammation and tissue damage and lower blood pressure in mice with pre-eclampsia induced by TLR3 or TLR7 activation. Injection of PLX-PAD cells on gestational day 14 significantly decreased systolic blood pressure by day 17 in TLR3-induced and TLR7-induced hypertensive mice (TLR3 144–111 mmHg; TLR7 145–106 mmHg; both P

Angiotensin II (AII)-dependent hypertension (AIIHTN) is associated with renal immune cell infiltration and inflammation. Lymphatic vessels drain interstitial fluid and traffic immune cells to draining lymph nodes; however, the role of renal lymphatics in AIIHTN is unknown. Our hypotheses were that: 1) renal lymphatic vessel density is increased in mice with AIIHTN, and 2) that further augmenting renal lymphatic vessels will prevent AIIHTN. Male and female mice were infused with AII (490 ng/kg/min) or saline for 2 or 3 weeks by subdermal osmotic mini pumps. Male and female mice with AIIHTN had markedly increased renal lymphatic vessel density compared to controls. AIIHTN males had significantly increased renal gene expression of the lymphatic vessel markers Lyve1, Pdpn, and Vegfr3, while Pdpn and the lymphangiogenic signal Vegfc were increased significantly in AIIHTN females. Kidneys of AIIHTN males had significantly increased F4/80+ macrophages at 2 weeks and F4/80+ macrophages and CD3e+ T cells at 3 weeks as determined by flow cytometry. Unlike in males, renal CD11c+ dendritic cells were increased significantly in females. Renal mRNA levels of the pro-inflammatory cyto/chemokines Tnfa , Il1b , Mcp1 , and Cxcl13 were elevated significantly in males at 2 and 3 weeks and in females at 3 weeks. To determine whether augmenting renal lymphatic vessels prior to AII infusion could prevent AIIHTN, we used transgenic mice that overexpress the lymphangiogenic signal VEGF-D only in the kidney under the control of doxycycline (KidVD+ mice) and thus exhibit renal-specific lymphangiogenesis. Doxycycline initiated 1 week prior to 3-week AII infusion prevented AIIHTN in KidVD+ mice while KidVD- mice still developed AIIHTN (Males SBP: 122±2 vs. 161±3 mmHg; pTnfa and Il1b were normalized in all KidVD+ mice. These data demonstrate that renal lymphatic vessel density is increased in AIIHTN and that genetically inducing renal lymphangiogenesis prior to AII infusion can prevent AIIHTN by reducing renal immune cells and inflammation.

Activated immune cell infiltration into organs contributes to the development and maintenance of hypertension. Studies targeting specific immune cell populations or reducing their inflammatory signalling have demonstrated a reduction in BP. Lymphatic vessels play a key role in immune cell trafficking and in resolving inflammation, but little is known about their role in hypertension. Studies from our laboratory and others suggest that inflammation-associated or induction of lymphangiogenesis is organ protective and anti-hypertensive. This review provides the basis for hypertension as a disease of chronic inflammation in various tissues and highlights how renal lymphangiogenesis is a novel regulator of kidney health and BP. LINKED ARTICLES: This article is part of a themed section on Immune Targets in Hypertension. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.12/issuetoc.

In humans and experimental animals, persistent immune system activation, accumulation of immune cells in the kidney, and subsequent inflammation plays an essential role in the development of hypertension (HTN). To reduce inflammation, lymphatic vessels drain extracellular fluid from the interstitium and traffic immune cells to draining lymph nodes. However, little is known about the connection between hypertension and renal lymphatic vessels. We hypothesized that renal lymphatic vessel density would increase in mice with L-NAME HTN and that genetically induced renal lymphangiogenesis would prevent this increase in blood pressure. L-NAME (0.5 mg/mL) was administered in the drinking water for two weeks and caused HTN (SBP: 153±3 vs. 103±3 mmHg; pVegfc , macrophage marker Adgre1 (F4/80), dendritic cell marker Cd11c , Th1 cell marker Tbx21 , and the pro-inflammatory cytokine Il6 . Blood pressure decreased after a two-week washout period following L-NAME (SBP: 113±2 mmHg) which was associated with a decrease in renal gene expression of Adgre1 (F4/80) and Cd11c , however renal lymphatic vessels remained dilated. To determine if augmenting renal lymphatic vessel density prior to L-NAME treatment would prevent HTN, we used transgenic mice that in response to doxycycline undergo kidney-specific VEGF-D overexpression (KidVD+ mice) and renal lymphangiogenesis. Doxycycline (200 mg/L) was administered in the drinking water of KidVD+ and KidVD- mice for four weeks with L-NAME being added during the final three weeks. Starting doxycycline one week prior to L-NAME prevented HTN in KidVD+ mice while slightly decreasing SBP in KidVD- mice (SBP: 112±4 vs. 134±2 mmHg; pRorc was decreased and the lymphatic chemokine markers Ccl21 and Ccl19 were increased significantly in KidVD+ mice. These data together demonstrate that L-NAME HTN can alter the size of renal lymphatic vessels and genetically augmenting renal lymphatic vessel density prior to L-NAME can prevent the development of HTN.

Salt-sensitive hypertension (SSHTN) is associated with renal immune cell infiltration and interstitial inflammation. Lymphatic vessels drain the interstitial compartment and traffic immune cells to draining lymph nodes; however little is known about the role of lymphatics and immune cell trafficking in the kidney during SSHTN. Our hypotheses were that renal lymphatic vessel density is increased in mice with SSHTN and that further augmenting renal lymphatic vessels will prevent SSHTN. SSHTN mice were made by administering L-NAME for two weeks, followed by a two week washout, and then were fed a 4% high salt diet for three weeks. Compared to control mice, mice with SSHTN (SBP: 103±3 vs. 136±2 mmHg; pLyve1 , the macrophage marker Adgre1 (F4/80), the Th1 cell marker Tbx21 , and the pro-inflammatory cytokine Il6 while expression of the immune cell-lymphatic chemokine receptor Ccr7 was decreased significantly. Mice solely fed a 4% salt diet for three weeks did not exhibit hypertension or increased renal lymphatic vessel density. To determine whether augmenting renal lymphatic vessels prior to the high salt diet could prevent SSHTN, we used transgenic mice that overexpress the lymphangiogenic signal VEGF-D only in the kidney under the control of doxycycline (KidVD+ mice) and thus exhibit renal lymphangiogenesis. Doxycycline initiated one week prior to the high salt diet prevented SSHTN in KidVD+ mice while having no effect on blood pressure in KidVD- mice (SBP: 117±4 vs. 139±5 mmHg; pTbx21 was decreased in KidVD+ mice while Ccr7 gene expression was increased significantly. These data demonstrate that renal lymphatic vessel density is increased in SSHTN and that augmenting renal lymphatic vessel density prior to a high salt diet can prevent SSHTN by improving renal immune cell exfiltration.

Excessive innate immune system activation and inflammation during pregnancy can lead to organ injury and dysfunction and preeclampsia (PE); however, the molecular mechanisms involved are unknown. We tested the hypothesis that Toll-like receptor (TLR) activation induces major histocompatibility complex (MHC) class II invariant chain peptide (CLIP) expression on immune cells, makes them pro-inflammatory, and are necessary to cause PE-like features in mice. Treatment with VG1177, a competitive antagonist peptide for CLIP in the groove of MHC class II, was able to both prevent and treat PE-like features in mice. We then determined that γ–δ T cells are critical for the development of PE-like features in mice since γ–δ T-cell knockout mice, like CLIP deficient mice, are resistant to developing PE-like features. Placentas from women with PE exhibit significantly increased levels of γ–δ T cells. These preclinical data demonstrate that CLIP expression and activated γ–δ T cells are responsible for the development of immunologic PE-like features and that temporarily antagonizing CLIP and/or γ–δ T cells may be a therapeutic strategy for PE.

Persistent immune system activation plays an important role in the development of various forms of hypertension. Activation of the innate immune system, inflammation, and subsequent adaptive immune system response causing end-organ injury and dysfunction ultimately leads to hypertension and its associated sequelae including coronary artery disease, heart failure, stroke, and chronic kidney disease. In this review, we will provide updates on the innate and adaptive immune cells involved in hypertension, the current understanding of how the immune system gets activated, and examine the recently discovered mechanisms involved in several forms of experimental hypertension.

Lymphatic vessels are vital for the trafficking of immune cells from the interstitium to draining lymph nodes during inflammation. Hypertension is associated with renal infiltration of activated immune cells and inflammation; however, it is unknown how renal lymphatic vessels change in hypertension. We hypothesized that renal macrophage infiltration and inflammation would cause increased lymphatic vessel density in hypertensive rats. Spontaneously hypertensive rats (SHR) that exhibit hypertension and renal injury (SHR-A3 strain) had significantly increased renal lymphatic vessel density and macrophages at 40 wk of age compared with Wistar-Kyoto (WKY) controls. SHR rats that exhibit hypertension but minimal renal injury (SHR-B2 strain) had significantly less renal lymphatic vessel density compared with WKY rats. The signals for lymphangiogenesis, VEGF-C and its receptor VEGF-R3, and proinflammatory cytokine genes increased significantly in the kidneys of SHR-A3 rats but not in SHR-B2 rats. Fischer 344 rats exhibit normal blood pressure but develop renal injury as they age. Kidneys from 24-mo- and/or 20-mo-old Fischer rats had significantly increased lymphatic vessel density, macrophage infiltration, VEGF-C and VEGF-R3 expression, and proinflammatory cytokine gene expression compared with 4-mo-old controls. These data together demonstrate that renal immune cell infiltration and inflammation cause lymphangiogenesis in hypertension- and aging-associated renal injury.

Objective: Inflammation is necessary for successfulpregnancy; however, excessive inflammation plays acentral role in the development of the pregnancy-specifichypertensive disorder preeclampsia. Numerous anti-inflammatory cytokines are decreased in women withpreeclampsia but the role of individual cytokines in bloodpressure regulation during pregnancy is unknown.Therefore, we examined whether the lack of the potentanti-inflammatory cytokine interleukin-4 (IL-4) would besufficient to elicit a preeclampsia-like syndrome in mice,and when coupled with immune system activation thatthese symptoms would be further augmented.Methods: Measures of splenic immune cells, placentalinflammation, blood pressure, endothelial function, andurinary protein excretion were performed in pregnant IL-4-deficient mice as well as in pregnant IL-4-deficient micetreated with the Toll-like receptor 3 agonistpolyinosinic:polycytidylic (poly I:C).Results: Pregnant IL-4-deficient mice exhibited alteredsplenic immune cell subsets, increased levels of pro-inflammatory cytokines, placental inflammation, mildhypertension, endothelial dysfunction, and proteinuriacompared to pregnant control mice. Compared topregnant control mice treated with poly I:C which exhibitpreeclampsia-like symptoms, poly I:C-treated pregnant IL-4-deficient mice exhibited a further increase in pro-inflammatory cytokine levels, which was associated withaugmented SBP and endothelial dysfunction.Conclusion: Collectively, these data show that theabsence of IL-4 is sufficient to induce mild preeclampsia-like symptoms in mice due to excessive inflammation.Thus, the anti-inflammatory effects of IL-4 are important inpreventing hypertension during pregnancy.Keywords: anti-inflammatory, cytokines, endothelialfunction, pregnancy, pro-inflammatory, SBPAbbreviations: ACh, acetylcholine; DAMPs, danger-associated molecular patterns; ICAM-1, intracellularadhesion molecule-1; IFNg, interferon g; IL, interleukin;PAMPs, pathogen-associated molecular patterns; polyI???C, polyinosinic???polycytidylic; SNP, sodiumnitroprusside; TGFb1, transforming growth factor b1; Th, Thelper type; TLR, Toll-like receptor; TNFa, tumor necrosisfactor a

Elevated levels of pro-inflammatory cytokine interleukin-17A (IL-17) are associated with hypertensive autoimmune diseases; however, the connection between IL-17 and hypertension is unknown. We hypothesized that IL-17 increases blood pressure by decreasing endothelial nitric oxide production. Methods and results Acute treatment of endothelial cells with IL-17 caused a significant increase in phosphorylation of the inhibitory endothelial nitric oxide (NO) synthase residue threonine 495 (eNOS Thr495). Of the kinases known to phosphor- ylate eNOS Thr495, only inhibition of Rho-kinase prevented the IL-17-induced increase. IL-17 caused a threefold increase in the Rho-kinase activator RhoA, and this was prevented by an IL-17 neutralizing antibody. In isolated mouse aortas, IL-17 significantly increased eNOS Thr495 phosphorylation, induced RhoA expression, and decreased NO-dependent relaxation responses, all of which were prevented by either an IL-17 neutralizing antibody or inhib- ition of Rho-kinase. In mice, IL-17 treatment for 1 week significantly increased systolic blood pressure and this was associated with decreased aortic NO-dependent relaxation responses, increased eNOS Thr495 phosphorylation, and increased RhoA expression. Inhibition of Rho-kinase prevented the hypertension caused by IL-17. Conclusion These data demonstrate that IL-17 activates RhoA/Rho-kinase leading to endothelial dysfunction and hypertension. Inhibitors of IL-17 or Rho-kinase may prove useful as anti-hypertensive drugs in IL-17-associated autoimmune

Hypertension is associated with immune system activation and inflammation. Renal infiltration of both innate and adaptive immune cells contributes to injury, dysfunction, and increased blood pressure. Activated immune cells that exit blood vessels into the interstitium then travel through lymphatic vessels to draining lymph nodes where they signal to other immune cells to increase the immune response. It is unknown how renal lymphatic vessels change in the context of hypertension, immune system activation, inflammation, and injury. We hypothesized that renal macrophage infiltration, inflammation, and injury would significantly increase lymphangiogenesis in various strains of rats. SHR rats that exhibit hypertension and renal injury (SHR-A3 strain) had significantly increased numbers of renal lymphatic vessels at 40 weeks of age compared to WKY controls (total of 3 fields of view: 52 ± 1 vs. 28 ± 1; p

Pin1 is a peptidyl prolyl cis-trans isomerase that only binds to and isomerizes phosphorylated serine/threonine-proline motifs, inducing conformational changes that alter target protein function and phosphorylation. We have shown previously that deficiency of another peptidyl prolyl isomerase, FK506 binding protein 12/12.6, alters endothelial NO synthase phosphorylation and causes endothelial dysfunction and hypertension. Endothelial NO synthase contains the Pin1 binding sequence at (p)serine 116-proline 117 and phosphorylation of endothelial NO synthase serine 116 inhibits NO production; however, whether Pin1 deficiency alters vascular function and blood pressure is unknown. We hypothesized that Pin1 isomerizes p-endothelial NO synthase serine 116, which enables dephosphorylation and stimulates NO production. Immunoprecipitation of endothelial NO synthase and probing for Pin1 in rat aortic endothelial cells confirmed the interaction between the two. Pin1 knockdown via small interfering RNA or inhibition by juglone increased endothelial NO synthase serine 116 phosphorylation and prevented vascular endothelial growth factor–induced serine 116 dephosphorylation in endothelial cells. Acute treatment of isolated mouse aortas with juglone increased endothelial NO synthase serine 116 phosphorylation and decreased NO production and relaxation responses. Mice treated with juglone for 2 weeks, as well as Pin1 knockout mice, exhibited increased aortic endothelial NO synthase serine 116 phosphorylation, endothelial dysfunction, and hypertension. These data demonstrate that Pin1 binds endothelial NO synthase and enables dephosphorylation of serine 116, which increases NO production and endothelium-dependent dilation, leading to blood pressure maintenance.

Preeclampsia may result from overactivation of the maternal immune system and is characterized by endothelial dysfunction and excessive inflammation. Given the importance of maternal immune system regulation and anti-inflammatory cytokines in normotensive pregnancies, we hypothesized that maternal immune system activation via Toll-like receptor 3 during pregnancy would cause preeclampsia-like symptoms in mice, which would be made worse by deficiency of the anti-inflammatory cytokine interleukin 10. The Toll-like receptor 3 agonist polyinosine-polycytidylic acid (poly I:C) caused hypertension, endothelial dysfunction, and proteinuria in mice only when pregnant. In the absence of poly I:C, pregnant interleukin 10 knockout mice exhibited a significant increase in systolic blood pressure, endothelial dysfunction, and serum proinflammatory cytokines, as well as aortic and placental platelet-endothelial cell adhesion molecule expression compared with pregnant wild-type mice. Deficiency of interleukin 10 further augmented these measures in poly I:C–treated pregnant mice. In addition, sera from poly I:C-treated pregnant wild-type mice significantly decreased relaxation responses and increased platelet-endothelial cell adhesion molecule expression in isolated aortas from nonpregnant wild-type mice, and these effects were augmented by sera from poly I:C-treated interleukin 10 knockout mice. Coincubation with recombinant interleukin 10 normalized relaxation responses and platelet-endothelial cell adhesion molecule expression in all of the groups. Collectively, Toll-like receptor 3 activation during pregnancy causes preeclampsia-like symptoms, which are exacerbated by the absence of interleukin 10. Exogenous interleukin 10 treatment had beneficial effects on endothelial function and may be beneficial in women with preeclampsia.

Hypertensive disorders of pregnancy are characterized by systemic and placental inflammation; however, treatment for these conditions has remained elusive. We tested whether administration of the anti-inflammatory cytokine interleukin-10 (IL-10) during pregnancy would attenuate the hypertension, endothelial dysfunction, proteinuria, and inflammation seen in pregnant DOCA/saline-treated (PDS) rats. Normal pregnant (NP) rats and PDS were given daily intraperitoneal injections of recombinant IL-10 from gestational day 13 until death on day 20. Systolic blood pressure, aortic endothelium-dependent relaxation responses, and urinary protein excretion were measured on days 13 and 20 of gestation. Fetal number and development, plasma endothelin-1 levels, serum and placental levels of IFNγ and IL-10, and aortic and placental levels of platelet endothelial cell adhesion molecule (PECAM) were assessed on gestational day 20. Systolic blood pressure, aortic endothelial dysfunction, and urinary protein excretion were significantly increased at gestational day 13 in PDS rats. However, all of these were restored to NP levels following IL-10 treatment in PDS rats. IL-10 treatment also significantly increased the number of pups per litter in PDS rats and did not further affect fetal development. The beneficial effects of IL-10 in PDS rats were likely mediated by the decreased plasma levels of endothelin-1, decreased levels of circulating and placental IFNγ, as well as decreased aortic and placental expression of PECAM. These data demonstrate that exogenous IL-10 can normalize blood pressure and endothelial function in pregnancy-induced hypertensive rats and may be beneficial in women with hypertensive disorders of pregnancy.

Background and Objectives. We have developed a rat model of preeclampsia which is based upon excessive volume expansion and includes hypertension, proteinuria and intrauterine growth restriction. In this model, the urinary excretion of the circulating steroid inhibitor of Na +/ K+ ATPase, marinobufagenin, is increased prior to the development of hypertension and proteinuria. An analogue of marinobufagenin, resibufogenin, successfully treats the hypertension and proteinuria. Methods. We administered resibufogenin early in pregnancy in this model, prior to the development of the syndrome. Results. We found that resibufogenin not only prevented the advent of hypertension and proteinuria, but also the development of intrauterine growth restriction. Discussion. These results may have relevance to the human condition.

BACKGROUND Preeclampsia (PE), a pregnancy-specific hypertensive syndrome, is one of the leading causes of premature births as well as fetal and maternal death. There is strong evidence that maternal immune system activation, of which Toll-like receptors (TLRs) play a major role, contributes to the development of PE. Viral infections, sensed by TLR3, are associated with hypertensive disorders of pregnancy. We tested the hypothesis that TLR3 activation during pregnancy would cause hypertension, endothelial dysfunction, proteinuria, and intrauterine growth restriction in normal pregnant rats. METHODS We treated pregnant and nonpregnant rats with the viral mimetic polyinosinic:polycytidylic acid (poly I:C) or vehicle every other day beginning at day 10 of gestation and measured systolic blood pressure, aortic vasodilation, urinary protein concentration, fetal growth, and serum and placental cytokine levels. RESULTS Pregnant rats treated with poly I:C displayed significantly elevated systolic blood pressures compared to pregnant rats and nonpregnant rats treated with poly I:C on day 18 of gestation. Poly I:C-treated pregnant rats also exhibited significantly decreased aortic vasodilation, significantly increased urinary protein concentrations, and had more malformed pups/litter. Additionally, poly I:C-treated rats exhibited a significant increase in placental TLR3 expression and proinflammatory/anti-inflammatory cytokine ratio compared to vehicle-treated rats. Poly I:C treatment of nonpregnant control rats had no effect on systolic blood pressure, aortic vasodilation, or urinary protein concentrations. CONCLUSION These findings demonstrate that sustained maternal immune system activation via TLR3 during pregnancy causes PE-like symptoms in rats and suggest that viral infection during pregnancy may contribute to the development of PE.