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1.  The effect of microvascular pattern alterations on network resistance in spontaneously hypertensive rats 
Structural microvascular rarefaction, defined by a loss of vessels, is a common characteristic of hypertension and has been associated with elevated microvascular resistance. However, determining the causal relationship between microvascular network structure and resistance requires a consideration of all pattern changes throughout a network. The objectives of this study were to determine whether microvascular rarefaction is associated with other network pattern alterations and to evaluate whether pattern alterations in hypertension necessarily contribute to increased microvascular resistance. Mesenteric tissues from age-matched (15–16 weeks) male spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats were harvested and immunolabeled for PECAM. SHR networks displayed a decreased microvascular area, arteriolar-venular (AV) length, number of AV branches, and number of capillary segments. In addition, SHR networks displayed increased AV connections per network compared to WKY networks. Based on network geometries, resistance per network was calculated using a computational model. For simulations with equal vessel diameter and with relative diameters based on reported intravital measurements, SHR microvascular network resistance was not elevated compared to the WKY level. Our results suggest that microvascular pattern alterations associated with hypertension are more complex than vessel loss, and that these combined alterations do not necessarily lead to elevated resistance.
PMCID: PMC3608097  PMID: 22562369
Microcirculation; Rarefaction; Hypertension; Mesentery; Resistance; SHR
2.  Matrix Metalloproteinase Activity Causes VEGFR-2 Cleavage and Microvascular Rarefaction in Rat Mesentery 
A complication of the spontaneously hypertensive rat (SHR) is microvascular rarefaction, defined by the loss of microvessels. However, the molecular mechanisms involved in this process remain incompletely identified. Recent work in our laboratory suggests that matrix metalloproteinases (MMPs) may play a role by cleavage of the vascular endothelial growth factor receptor 2 (VEGFR-2). In order to further delineate the role for MMPs in microvascular rarefaction, the objective of the current study was to examine the relationship in the same tissue between MMP activity, VEGFR-2 cleavage and rarefaction. Using an in-vivo microzymographic technique we show significantly enhanced levels of MMP-1, -1/-9, -7, and -8 activities, but not MMP-2 and-3 activities, along mesenteric microvessels of the SHR compared to its normotensive control, Wistar Kyoto (WKY) rat. Based on immunohistochemical methods, the SHR exhibited a decreased labeling of the extracellular, but not the intracellular, domain of VEGFR-2 along mesenteric microvessels. Chronic MMP inhibition served to attenuate VEGFR-2 cleavage and microvascular network rarefaction in the SHR mesentery. These results spatially link MMP-induced VEGFR-2 cleavage and rarefaction in the mesentery of the SHR and thus support the hypothesis that MMPs serve as regulators of microvascular dysfunction in hypertension.
PMCID: PMC3081385  PMID: 21418372
Spontaneously Hypertensive Rat; Wister Kyoto rat; capillary; arteriole; venule; microzymography; matrix metalloproteinase inhibition
Elevated blood pressure during hypertension has been associated with microvascular rarefaction defined by loss of microvessels. However, whether rarefaction is a result of impaired angiogenesis remains unclear. The objective of this study was to compare angiogenesis across the time course of mesenteric microvascular network remodeling in adult spontaneously hypertensive versus normotensive rats. Angiogenic responses in 15–16-week-old SHR and Wistar rats at 0, 3, 5, 10 or 25 days post 20 minute exteriorization of the mesentery were quantified. Consistent with the phenomenon of rarefaction, vascularized area in unstimulated SHR was decreased compared to Wistar. By 25 days, SHR vascular area had increased to the Wistar level and vascular length density and capillary sprouting were comparable. At 3 and 5 days, SHR and Wistar tissues displayed an increase in the capillary sprouting and vascular density relative to their unstimulated controls. At 10 days, capillary sprouting in the SHR remained elevated. The percent change in vascular density was elevated in the SHR compared to the Wistar group at 3 and 5 days and by 25 days the rate of change was more negative. Our results suggest that SHR networks undergo an increased rate of growth followed by an increased rate of pruning.
PMCID: PMC3176994  PMID: 21627712
Hypertension; Angiogenesis; Spontaneously Hypertensive Rat; Microcirculation; Mesentery
4.  Sex‐specific alterations in NOS regulation of vascular function in aorta and mesenteric arteries from spontaneously hypertensive rats compared to Wistar Kyoto rats 
Physiological Reports  2014;2(8):e12125.
The present study tested the hypothesis that spontaneously hypertensive rats (SHR) have impaired nitric oxide synthase (NOS)‐mediated regulation of vascular function versus Wistar‐Kyoto rats (WKY). Aorta and small mesenteric arteries were studied from male and female SHR (M SHR and F SHR) and WKY (M WKY and F WKY). Phenylephrine (PE)‐induced vasoconstriction was greater in aorta of M SHR versus all others (P < 0.05); there were neither sex nor strain differences in PE contraction in mesenteric arteries. The NOS inhibitor l‐Nitro‐Arginine Methyl Ester (l‐NAME) increased PE‐induced vasoconstriction in all rats, although the increase was the least in male SHR (P < 0.05), revealing a blunted vasoconstrictor buffering capacity of NOS. l‐NAME increased sensitivity to PE‐induced constriction only in mesenteric arteries of SHR, although, the maximal percent increase in contraction was comparable among groups. ACh‐induced relaxation was also less in aorta from M SHR versus all others (P < 0.05). ACh relaxation was comparable among groups in mesenteric arteries, although SHR exhibited a greater NOS component to ACh‐induced relaxation than WKY. To gain mechanistic insight into sex and strain differences in vascular function, NOS activity and NOS3 protein expression were measured. Aortic NOS activity was comparable between groups and M SHR had greater NOS3 expression than M WKY. In contrast, although vascular function was largely maintained in mesenteric arteries of SHR, NOS activity was less in SHR versus WKY. In conclusion, M SHR exhibit a decrease in NOS regulation of vascular function compared to F SHR and WKY, although this is not mediated by decreases in NOS activity and/or expression.
The present study tested the hypothesis that spontaneously hypertensive rats (SHR) have impaired nitric oxide synthase (NOS)‐mediated regulation of vascular function versus Wistar‐Kyoto rats (WKY). Aorta and small mesenteric arteries were studied from male and female SHR and WKY. Male SHR showed a decreased NOS regulation of vascular function compared to F SHR and WKY, although this was not mediated by decreases in NOS activity and/or expression.
PMCID: PMC4246578  PMID: 25168874
NOS activity; NOS expression; phenylephrine; SHR
5.  Blood pressure development of the spontaneously hypertensive rat after concurrent manipulations of dietary Ca2+ and Na+. Relation to intestinal Ca2+ fluxes. 
Journal of Clinical Investigation  1985;76(3):1147-1154.
The blood pressure of the spontaneously hypertensive rat (SHR) is influenced by the Ca2+ content of its diet. As the SHR's greater dependence on dietary calcium may reflect a defect in intestinal calcium absorption, we measured in vitro unidirectional Ca2+ flux (J) in the duodenum-jejunum (four segments each) of the SHR (n = 6) and the normotensive Wistar-Kyoto rat (WKY; n = 6) by a modified Ussing apparatus. Because of the known and postulated interactions between Ca2+ and Na+ in both intestinal and vascular tissue, we assessed in vivo the influence of a concurrent manipulation of Na+ intake (three levels: 0.25%, 0.45%, and 1.0%) on the blood pressure development of SHRs (n = 35) and WKYs (n = 35), between 6 and 20 wk of age, exposed to three levels of dietary calcium (0.1, 1.0, and 2%). Net calcium flux (Jnet) (mean +/- SEM) was significantly (P less than 0.01) lower in the SHR (-2.8 +/- 6.3 nmol/cm2 X h) than in the WKY (34.6 +/- 8.8 nmol/cm2 X h). The SHR's decreased Jnet resulted from a significantly (P less than 0.03) lower mucosa-to-serosa flux (Jm-s) in the SHR (41.0 +/- 5.6 nmol/cm2 X h) compared with the Jm-s of the WKY (70.1 +/- 9.1 nmol/cm2 X h). Serosa-to-mucosa flux for calcium did not differ between the SHR (43.8 +/- 6.6 nmol/cm2 X h) and the WKY (35.5 +/- 8.0 nmol/cm2 X h). The SHR's decreased (P less than 0.002) Jm-s was confirmed by additional measurements in SHRs and WKYs. Jm-s was 36.2 +/- 3.7 nmol/cm2 X h in the SHRs (n = 11) and 64.4 +/- 6.7 nmol/cm2 X h in the WKYs (n = 9). The provision of an increased dietary Ca2+ (2% by weight) and increased Na+ (1%) to the SHR prevented the emergence of hypertension (P less than 0.001) (mean +/- SEM systolic blood pressure at 20 wk of age; 135 +/- 5 mmHg for the 2% Ca2+, 1% Na+ SHR vs. 164 +/- 2 mmHg for the control diet SHR). Ca2+ (0.1%) and Na+ (0.25%) restriction accelerated the SHR's hypertension (192 +/- 2 mmHg) (P less than 0.001) and was associated with higher pressures in the WKY (146 +/- 4 mmHg in the restricted WKY vs. 134 +/- 4 mmHg in the control WKY). In a parallel group of 24 SHRs and 24 WKYs fed one of three diets (2% Ca2+/1% Na+; 1% Ca2+/0.45% Na+; or 0.1% Ca2+/0.25% Na+), the heart (P < 0.05) and kidney (P = 0.08) weight of the SHRs varied depending on the diet at 20 wk of age. Low Ca2+ and Na+ intake was associated with increased heart weight (1.6+/-0.9 g) compared with the normal diet for SHR (1.51+/-0.07 g). Increased Ca2+ and Na+ intake was associated with a significantly (P = 0.05) lower heart weight in the SHR (1.37+/-0.03 g) and in the WKY (1.35+/-0.06 g) compared with their normal diet controls. These findings show one mechanism for the SHR's depressor response to supplemental dietary Ca2+ and, in part, explain the sodium dependence of calcium's cardiovascular protective effect.
PMCID: PMC424009  PMID: 4044829
6.  Molecular basis for impaired collateral artery growth in the spontaneously hypertensive rat: insight from microarray analysis 
Physiological Reports  2013;1(2):e0005.
Analysis of global gene expression in mesenteric control and collateral arteries was used to investigate potential molecules, pathways, and mechanisms responsible for impaired collateral growth in the Spontaneously Hypertensive Rat (SHR). A fundamental difference was observed in overall gene expression pattern in SHR versus Wistar Kyoto (WKY) collaterals; only 6% of genes altered in collaterals were similar between rat strains. Ingenuity® Pathway Analysis (IPA) identified major differences between WKY and SHR in networks and biological functions related to cell growth and proliferation and gene expression. In SHR control arteries, several mechano-sensitive and redox-dependent transcription regulators were downregulated including JUN (−5.2×, P = 0.02), EGR1 (−4.1×, P = 0.01), and NFĸB1 (−1.95×, P = 0.04). Predicted binding sites for NFĸB and AP-1 were present in genes altered in WKY but not SHR collaterals. Immunostaining showed increased NFĸB nuclear translocation in collateral arteries of WKY and apocynin-treated SHR, but not in untreated SHR. siRNA for the p65 subunit suppressed collateral growth in WKY, confirming a functional role of NFkB. Canonical pathways identified by IPA in WKY but not SHR included nitric oxide and renin–angiotensin system signaling. The angiotensin type 1 receptor (AGTR1) exhibited upregulation in WKY collaterals, but downregulation in SHR; pharmacological blockade of AGTR1 with losartan prevented collateral luminal expansion in WKY. Together, these results suggest that collateral growth impairment results from an abnormality in a fundamental regulatory mechanism that occurs at a level between signal transduction and gene transcription and implicate redox-dependent modulation of mechano-sensitive transcription factors such as NFĸB as a potential mechanism.
PMCID: PMC3831906  PMID: 24303120
Arteriogenesis; collateral gene expression; microarray analysis; peripheral vascular disease
7.  Upregulated TRPC3 and Downregulated TRPC1 Channel Expression during Hypertension is Associated with Increased Vascular Contractility in Rat 
Transient receptor potential (TRP) C1 and C3 (TRPC1 and TRPC3) are expressed in vascular smooth muscle cells and are thought to be involved in vascular contractility. In the present study, we determined the effect of systemic hypertension on TRPC1/TRPC3 channel expression and vascular contractility in rat carotid artery (CA). CA were studied from male spontaneously hypertensive rats (SHR), Wistar-Kyoto (WKY), and Long Evans (LE) rats. TRPC1/3 expression was determined by RT-PCR and Western blot. TRP channel function was evaluated by whole-cell patch clamp, using UTP (60 μM) to stimulate TRPC1/3 channels. Contractions of endothelium-denuded CA segments to UTP (1–300 μM) and phenylephrine (Phe; 0.1 nM–10 μM) were measured in an isometric tension bath. TRPC1 and TRPC3 mRNA was present in CA of both WKY and SHR. Western blot demonstrated 3.1 ± 1.2 times greater TRPC3 expression and 0.5 ± 0.2 times TRPC1 in SHR versus WKY CA. Isolated CA showed potentiated contraction to UTP in the SHR versus WKY. Activation of voltage-dependent Ca2+ channels (VDCC) in UTP-mediated constriction only occurred in SHR CA. Contraction to Phe was unaltered between WKY and SHR CA and involved equal significant VDCC activation in both groups. Patch clamp demonstrated that the UTP-stimulated current (Iutp) was greater in SHR compared to the normotensive WKY and LE rats with peak Iutp (at −110 mV) of −63 ± 24 pA compared to −25 ± 4 pA, respectively. We demonstrate that UTP-mediated but not Phe-mediated constrictions are potentiated in the CA during hypertension. Expression of TRPC1 is decreased whereas TRPC3 is increased in SHR CA. Interestingly, VDCC activation only contributes to UTP-mediated contraction of SHR CAs whereas it contributes substantially and equally in Phe-mediated contraction. We speculate that the alteration of TRPC channel expression in hypertension leads to greater smooth muscle depolarization, VDCC activation, and vascular contractility in the UTP (but not Phe) signaling pathway.
PMCID: PMC3143733  PMID: 21811471
calcium; carotid artery; hypertension; transient receptor potential channel; vascular smooth muscle
8.  Hypertension Is Associated with Marked Alterations in Sphingolipid Biology: A Potential Role for Ceramide 
PLoS ONE  2011;6(7):e21817.
Hypertension is, amongst others, characterized by endothelial dysfunction and vascular remodeling. As sphingolipids have been implicated in both the regulation of vascular contractility and growth, we investigated whether sphingolipid biology is altered in hypertension and whether this is reflected in altered vascular function.
Methods and Findings
In isolated carotid arteries from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats, shifting the ceramide/S1P ratio towards ceramide dominance by administration of a sphingosine kinase inhibitor (dimethylsphingosine) or exogenous application of sphingomyelinase, induced marked endothelium-dependent contractions in SHR vessels (DMS: 1.4±0.4 and SMase: 2.1±0.1 mN/mm; n = 10), that were virtually absent in WKY vessels (DMS: 0.0±0.0 and SMase: 0.6±0.1 mN/mm; n = 9, p<0.05). Imaging mass spectrometry and immunohistochemistry indicated that these contractions were most likely mediated by ceramide and dependent on iPLA2, cyclooxygenase-1 and thromboxane synthase. Expression levels of these enzymes were higher in SHR vessels. In concurrence, infusion of dimethylsphingosine caused a marked rise in blood pressure in anesthetized SHR (42±4%; n = 7), but not in WKY (−12±10%; n = 6). Lipidomics analysis by mass spectrometry, revealed elevated levels of ceramide in arterial tissue of SHR compared to WKY (691±42 vs. 419±27 pmol, n = 3–5 respectively, p<0.05). These pronounced alterations in SHR sphingolipid biology are also reflected in increased plasma ceramide levels (513±19 pmol WKY vs. 645±25 pmol SHR, n = 6–12, p<0.05). Interestingly, we observed similar increases in ceramide levels (correlating with hypertension grade) in plasma from humans with essential hypertension (185±8 pmol vs. 252±23 pmol; n = 18 normotensive vs. n = 19 hypertensive patients, p<0.05).
Hypertension is associated with marked alterations in vascular sphingolipid biology such as elevated ceramide levels and signaling, that contribute to increased vascular tone.
PMCID: PMC3139577  PMID: 21818267
9.  Heterogeneity in Arterial Remodeling among Sublines of Spontaneously Hypertensive Rats 
PLoS ONE  2014;9(9):e107998.
Spontaneously hypertensive rats (SHR) have been used frequently as a model for human essential hypertension. However, both the SHR and its normotensive control, the Wistar Kyoto rat (WKY), consist of genetically different sublines. We tested the hypothesis that the pathophysiology of vascular remodeling in hypertension differs among rat sublines.
Methods and Results
We studied mesenteric resistance arteries of WKY and SHR from three different sources, at 6 weeks and 5 months of age. Sublines of WKY and SHR showed differences in blood pressure, body weight, vascular remodeling, endothelial function, and vessel ultrastructure. Common features in small mesenteric arteries from SHR were an increase in wall thickness, wall-to-lumen ratio, and internal elastic lamina thickness.
Endothelial dysfunction, vascular stiffening, and inward remodeling of small mesenteric arteries are not common features of hypertension, but are subline-dependent. Differences in genetic background associate with different types of vascular remodeling in hypertensive rats.
PMCID: PMC4175999  PMID: 25251068
10.  Electroacupuncture Delays Hypertension Development through Enhancing NO/NOS Activity in Spontaneously Hypertensive Rats 
Using spontaneously hypertensive rats (SHR), this study investigated whether electroacupuncture (EA) could reduce early stage hypertension by examining nitric oxide (NO) levels in plasma and nitric oxide synthase (NOS) levels in the mesenteric resistance artery. EA was applied to the acupuncture point Governor Vessel 20 (GV20) or to a non-acupuncture point in the tail twice weekly for 3 weeks under anesthesia. In conscious SHR and normotensive Wistar Kyoto (WKY) rats, blood pressure was determined the day after EA treatment by the tail-cuff method. We measured plasma NO concentration, and evaluated endothelial NO syntheses (eNOS) and neuronal NOS (nNOS) protein expression in the mesenteric artery. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were lower after 3 weeks of GV20 treatment than EA at non-acupuncture point and no treatment control in SHR. nNOS expression by EA was significantly different between both WKY and no treatment SHR control, and EA at GV20 in SHR. eNOS expression was significantly high in EA at GV 20 compared with no treatment control. In conclusion, EA could attenuate the blood pressure elevation of SHR, along with enhancing NO/NOS activity in the mesenteric artery in SHR.
PMCID: PMC3094701  PMID: 18955367
11.  Regulation of 3’,5’-cAMP in Preglomerular Smooth Muscle and Endothelial Cells From Genetically-Hypertensive Rats 
Hypertension  2010;56(6):1096-1101.
Our previous studies show that inhibition of phosphodiesterase 4 (PDE4) augments agonist-induced renovascular 3’,5’-cAMP secretion more in isolated, perfused kidneys from spontaneously hypertensive rats (SHR) versus Wistar-Kyoto normotensive rats (WKY); however, whether this is due to PDE4 inhibition in renovascular smooth muscle cells or endothelial cells is unknown. Therefore, we examined the effects of 3-isobutyl-1-methylxanthine (broad-spectrum PDE inhibitor) and RO 20–1724 (selective PDE4 inhibitor) on isoproterenol-induced 3’,5’-cAMP levels in cultured WKY and SHR preglomerular vascular smooth muscle and endothelial cells. 3-Isobutyl-1-methylxanthine and RO 20–1724 augmented isoproterenol-induced 3’,5’-cAMP levels similarly in WKY versus SHR endothelial cells. In contrast, 3-isobutyl-1-methylxanthine and RO 20–1724 augmented isoproterenol-induced 3’,5’-cAMP levels significantly more in SHR, compared with WKY, smooth muscle cells (p<0.0001). In both cell types from both rat strains, mRNA levels for the PDE4B subtype exceeded levels for the PDE4A, PDE4C and PDE4D subtypes, and siRNA knockdown of PDE4B mRNA in SHR smooth muscle cells increased isoproterenol-induced 3’,5’-cAMP. mRNA levels for the PDE4B2 variant exceeded levels for the PDE4B1, PDE4B3, PDE4B4 and PDE4B5 variants. In vivo, infusions of RO 20–1724 increased the urinary excretion of 3’,5’-cAMP more in SHR than WKY (p=0.0211). We conclude that: 1) the greater effect of PDE4 inhibition on renovascular 3’,5’-cAMP is mediated by inhibition of PDE4 in renovascular smooth muscle cells, not endothelial cells; 2) the major PDE4 subtype in both renovascular smooth muscle and endothelial cells is PDE4B with variant PDE4B2 likely being dominant; and 3) inhibition of PDE4 in vivo increases renal 3’,5’-cAMP levels more in genetically-hypertensive rats.
PMCID: PMC3011225  PMID: 20975032
3’,5’-Cyclic AMP; Phosphodiesterase 4; Phosphodiesterase 4B; Phosphodiesterase 4B2; Isoproterenol; Spontaneously Hypertensive Rats; Wistar-Kyoto Rats
12.  Longitudinal Evaluation of Left Ventricular Substrate Metabolism, Perfusion, and Dysfunction in the Spontaneously Hypertensive Rat Model of Hypertrophy Using Small-Animal PET/CT Imaging 
Myocardial metabolic and perfusion imaging is a vital tool for understanding the physiologic consequences of heart failure. We used PET imaging to examine the longitudinal kinetics of 18F-FDG and 14(R,S)-18F-fluoro-6-thia-heptadecanoic acid (18F-FTHA) as analogs of glucose and fatty acid (FA) to quantify metabolic substrate shifts with the spontaneously hypertensive rat (SHR) as a model of left ventricular hypertrophy (LVH) and failure. Myocardial perfusion and left ventricular function were also investigated using a newly developed radiotracer 18F-fluorodihydrorotenol (18F-FDHROL).
Longitudinal dynamic electrocardiogram-gated small-animal PET/CT studies were performed with 8 SHR and 8 normotensive Wistar-Kyoto (WKY) rats over their life cycle. We determined the myocardial influx rate constant for 18F-FDG and 18F-FTHA (KiFDG and KiFTHA, respectively) and the wash-in rate constant for 18F-FDHROL (K1FDHROL). 18F-FDHROL data were also used to quantify left ventricular ejection fraction (LVEF) and end-diastolic volume (EDV). Blood samples were drawn to independently measure plasma concentrations of glucose, insulin, and free fatty acids (FFAs).
KiFDG and KiFTHA were higher in SHRs than WKY rats (P < 3 × 10−8 and 0.005, respectively) independent of age. A decrease in KiFDG with age was evident when models were combined (P = 0.034). The SHR exhibited higher K1FDHROL (P < 5 × 10−6) than the control, with no age-dependent trends in either model (P = 0.058). Glucose plasma concentrations were lower in SHRs than controls (P < 6 × 10−12), with an age-dependent rise for WKY rats (P < 2 × 10−5). Insulin plasma concentrations were higher in SHRs than controls (P < 3 × 10−3), with an age-dependent decrease when models were combined (P = 0.046). FFA levels were similar between models (P = 0.374), but an increase with age was evident only in SHR (P < 7 × 10−6).
The SHR exhibited alterations in myocardial substrate use at 8 mo characterized by increased glucose and FA utilizations. At 20 mo, the SHR had LVH characterized by decreased LVEF and increased EDV, while simultaneously sustaining higher glucose and similar FA utilizations (compared with WKY rats), which indicates maladaptation of energy substrates in the failing heart. Elevated K1FDHROL in the SHR may reflect elevated oxygen consumption and decreased capillary density in the hypertrophied heart. From our findings, metabolic changes appear to precede mechanical changes of LVH progression in the SHR model.
PMCID: PMC4000452  PMID: 24092939
spontaneously hypertensive rat; myocardial substrate metabolism; myocardial perfusion; 18F-fluorodihydrorotenol; 18F-fluoro-6-thia-heptadecanoic acid
13.  Relaxin Suppresses Atrial Fibrillation by Reversing Fibrosis and Myocyte Hypertrophy, and Increasing Conduction Velocity and Sodium Current in Spontaneously Hypertensive Rat Hearts 
Circulation research  2013;113(3):313-321.
Atrial fibrillation (AF) contributes significantly to morbidity and mortality in elderly and hypertensive patients and has been correlated to enhanced atrial fibrosis. Despite a lack of direct evidence that fibrosis causes AF, reversal of fibrosis is considered as a plausible therapy.
To evaluate the efficacy of the anti-fibrotic hormone relaxin (RLX) at suppressing AF in spontaneously hypertensive rats (SHR).
Methods and Results
Normotensive Wistar Kyoto (WKY) and SHR were treated for 2-weeks with vehicle (WKY+V and SHR+V), or RLX (0.4mg/kg/day, SHR+RLX) using implantable mini-pumps. Hearts were perfused, mapped optically to analyze action potential durations (APDs), intracellular Ca2+-transients, restitution kinetics (RK) and tested for AF vulnerability. SHR hearts had slower conduction velocity (CV) (p< 0.01 vs. WKY), steeper CV RKs, greater collagen deposition, higher levels of transcripts for TGFβ, metalloproteinase-2, metalloproteinase-9, collagen I/III and reduced connexin-43 phosphorylation (p< 0.05 vs. WKY). Programmed stimulation triggered sustained AF in SHR (n=5/5), SHR+V (n=4/4) but not in WKY (n=0/5) and SHR+RLX (n=1/8, p<0.01). RLX treatment reversed the transcripts for fibrosis, flattened CV-RK, reduced APD90, increased CV (p<0.01) and reversed atrial hypertrophy (p<0.05). Independent of anti-fibrotic actions, RLX (0.1µM) increased Na+-current density, INa (~2-fold in 48-hours) in human cardiomyocytes derived from iPSCs (n=18/18, p<0.01).
RLX-treatment suppressed AF in SHR hearts by increasing CV from a combination of reversal of fibrosis and hypertrophy and increasing INa. The study provides compelling evidence that RLX may provide a novel therapy to manage AF in humans by reversing fibrosis, and hypertrophy and modulating cardiac ionic currents.
PMCID: PMC3774019  PMID: 23748429
Relaxin; atrial fibrillation; fibrosis; hypertrophy; INa upregulation; optical mapping; spontanteously hypertensiver rats
14.  Alterations in vasomotor systems and mechanics of resistance-sized mesenteric arteries from SHR and WKY male rats following in vivo testosterone manipulation 
Testosterone (T) and the sympathetic nervous system each contribute to the pathology of hypertension. Altered blood vessel reactivity is also associated with the pathology of high blood pressure. The purpose of this study was to examine the effects of T manipulation in the regulation of resistance-sized blood vessel reactivity.
Adult spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) male rats at 8 weeks of age were used. The rats were divided into groups consisting of gonadally intact controls (CONT), castrate with sham implant (CAST) and castrate with T implant (CAST + T) (n = 6 to 12 per group). Following a short-term period of T treatment (approximately 4 weeks), plasma norepinephrine (NE) and plasma T were assessed by performing high-performance liquid chromatography and RIA, respectively. Resistance-sized mesenteric artery reactivity was assessed on a pressurized arteriograph for myogenic reactivity (MYO), phenylephrine (PE) responsiveness and passive structural mechanics.
SHR and WKY males exhibited similar physiological trends in T manipulation, with castration significantly lowering plasma T and NE and T replacement significantly increasing plasma T and NE. T manipulation in general resulted in significant alterations in MYO of second-order mesenteric arteries, with T replacement decreasing MYO in SHR (P < 0.05) compared to CONT, T replacement increasing MYO, and CAST decreasing MYO in WKY rats (P < 0.001) compared to CONT rats. Additionally, PE-induced constriction was significantly altered in both strains following T treatment, with the effective concentration of PE to constrict the vessel to 50% of the total diameter significantly increased in the CAST + T SHR compared to CONT (P < 0.05). Comparisons of passive structural mechanics between SHR and WKY treatment groups indicated in SHR a significantly increased wall-to-lumen ratio and decreased circumferential wall stress compared to WKY treatment groups.
These data suggest that T and NE are involved in a complex interaction with both myogenic reactivity and structural alterations of resistance-sized blood vessels and that these factors likely contribute to the development and maintenance of hypertension.
PMCID: PMC3264497  PMID: 22214247
testosterone; norepinephrine; blood vessel; myogenic
15.  Sural nerve involvement in experimental hypertension: morphology and morphometry in male and female normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) 
BMC Neuroscience  2012;13:24.
The sural nerve has been widely investigated in experimental models of neuropathies but information about its involvement in hypertension was not yet explored. The aim of the present study was to compare the morphological and morphometric aspects of different segments of the sural nerve in male and female spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. Rats aged 20 weeks (N = 6 in each group) were investigated. After arterial pressure and heart rate recordings in anesthetized animals, right and left sural nerves were removed and prepared for epoxy resin embedding and light microscopy. Morphometric analysis was performed with the aid of computer software, and took into consideration the fascicle area and diameter, as well as myelinated fiber number, density, area and diameter.
Significant differences were observed for the myelinated fiber number and density, comparing different genders of WKY and SHR. Also, significant differences for the morphological (thickening of the endoneural blood vessel walls and lumen reduction) and morphometric (myelinated fibers diameter and G ratio) parameters of myelinated fibers were identified. Morphological exam of the myelinated fibers suggested the presence of a neuropathy due to hypertension in both SHR genders.
These results indicate that hypertension altered important morphometric parameters related to nerve conduction of sural nerve in hypertensive animals. Moreover the comparison between males and females of WKY and SHR allows the conclusion that the morphological and morphometric parameters of sural nerve are not gender related. The morphometric approach confirmed the presence of neuropathy, mainly associated to the small myelinated fibers. In conclusion, the present study collected evidences that the high blood pressure in SHR is affecting the sural nerve myelinated fibers.
PMCID: PMC3350456  PMID: 22380617
Sural nerve; Morphometry; Myelinated fibers; Spontaneously Hypertensive Rat; Gender differences
16.  An Elevation in Physical Coupling of Type 1 IP3 Receptors to TRPC3 Channels Constricts Mesenteric Arteries in Genetic Hypertension 
Hypertension  2012;60(5):1213-1219.
Hypertension is associated with an elevation in agonist-induced vasoconstriction, but mechanisms involved require further investigation. Many vasoconstrictors bind to phospholipase C-coupled receptors, leading to an elevation in inositol 1,4,5-trisphosphate (IP3) that activates sarcoplasmic reticulum (SR) IP3 receptors (IP3Rs). In cerebral artery myocytes, IP3Rs release SR Ca2+ and can physically couple to canonical transient receptor potential 3 (TRPC3) channels in a caveolin-1-containing macromolecular complex, leading to cation current (ICat) activation that stimulates vasoconstriction. Here, we investigated mechanisms by which IP3Rs control vascular contractility in systemic arteries and IP3R involvement in elevated agonist-induced vasoconstriction during hypertension. Total and plasma membrane-localized TRPC3 protein was ~2.7- and 2-fold higher in mesenteric arteries of hypertensive spontaneously hypertensive rats (SHR) than in Wistar-Kyoto (WKY) rat controls, respectively. In contrast, IP3R1, TRPC1, TRPC6, and caveolin-1 expression was similar. TRPC3 expression was also similar in arteries of pre-hypertensive SHR and WKY rats. Control, IP3- and endothelin-1 (ET-1)-induced FRET between IP3R1 and TRPC3 was higher in hypertensive SHR than WKY myocytes. IP3-induced ICat was ~3-fold larger in SHR myocytes. Pyr3, a selective TRPC3 channel blocker, and CIRBP-TAT, an IP3R-TRP physical coupling inhibitor, reduced IP3-induced ICat and ET-1-induced vasoconstriction more in SHR than WKY myocytes and arteries. Thapsigargin, a SR Ca2+-ATPase blocker, did not alter ET-1-stimulated vasoconstriction in SHR or WKY arteries. These data indicate that ET-1 stimulates physical coupling of IP3R1 to TRPC3 channels in mesenteric artery myocytes, leading to vasoconstriction. Furthermore, an elevation in IP3R1 to TRPC3 channel molecular coupling augments ET-1-induced vasoconstriction during hypertension.
PMCID: PMC3632264  PMID: 23045459
Hypertension; vasoconstriction; TRPC channels; IP3 receptors; endothelin-1
17.  Role of Complement 3a in the Synthetic Phenotype and Angiotensin II-Production in Vascular Smooth Muscle Cells From Spontaneously Hypertensive Rats 
American Journal of Hypertension  2011;25(3):284-289.
Spontaneously hypertensive rats (SHR)-derived vascular smooth muscle cells (VSMCs) show exaggerated growth with a synthetic phenotype and angiotensin II (Ang II)-production. To evaluate the contribution of complement 3 (C3) or C3a toward these abnormalities in SHR, we examined effects of a C3a receptor inhibitor on proliferation, phenotype, and Ang II-production in VSMCs from SHR and Wistar–Kyoto (WKY) rats.
Expression of pre-pro-C3 messenger RNA (mRNA) and C3 protein was evaluated by reverse transcription-PCR and western blot analyses, and C3a receptor mRNA was evaluated by reverse transcription-PCR analysis in quiescent VSMCs from SHR and WKY rats. We examined the effects of the C3a inhibitor, SB290157, on proliferation and the expression of phenotype-marker and Krueppel-like factor 5 (KLF-5) mRNAs in VSMCs from SHR and WKY rats. We examined effects of C3a receptor inhibitor, SB290157, on Ang II-production in conditioned medium of VSMCs from SHR and WKY rats by a radioimmunoassay.
Expression of pre-pro-C3 mRNA and C3 protein was significantly higher in SHR VSMCs than WKY VSMCs. SB290157 significantly inhibited proliferation of VSMCs from SHR, but not in cells from WKY rats. Relative to WKY VSMCs, SB290157 significantly increased the low expression of SM22α mRNA and decreased the high expression of osteopontin mRNA in SHR VSMCs. SB290157 significantly decreased the high expression of KLF-5 and Ang II-production in VSMCs from SHR, but not in cells from WKY rats.
C3a induces exaggerated growth, a synthetic phenotype and Ang II-production in SHR-derived VSMCs. C3a may be primarily involved in cardiovascular remodeling in hypertension.
PMCID: PMC3280391  PMID: 22089112
angiotensin II; blood pressure; complement 3; hypertension; Kruppel-like factor 5; phenotype; proliferation; spontaneously hypertensive rat; vascular smooth muscle cell
18.  Cross-fostering does not alter the neurochemistry or behavior of spontaneously hypertensive rats 
Attention-deficit/hyperactivity disorder (ADHD) is a highly heritable developmental disorder resulting from complex gene-gene and gene-environment interactions. The most widely used animal model, the spontaneously hypertensive rat (SHR), displays the major symptoms of ADHD (deficits in attention, impulsivity and hyperactivity) and has a disturbance in the noradrenergic system when compared to control Wistar-Kyoto rats (WKY). The aim of the present study was to determine whether the ADHD-like characteristics of SHR were purely genetically determined or dependent on the gene-environment interaction provided by the SHR dam.
SHR/NCrl (Charles River, USA), WKY/NCrl (Charles River, USA) and Sprague Dawley rats (SD/Hsd, Harlan, UK) were bred at the University of Cape Town. Rat pups were cross-fostered on postnatal day 2 (PND 2). Control rats remained with their birth mothers to serve as a reference for their particular strain phenotype. Behavior in the open-field and the elevated-plus maze was assessed between PND 29 and 33. Two days later, rats were decapitated and glutamate-stimulated release of [3H]norepinephrine was determined in prefrontal cortex and hippocampal slices.
There was no significant effect of "strain of dam" but there was a significant effect of "pup strain" on all parameters investigated. SHR pups travelled a greater distance in the open field, spent a longer period of time in the inner zone and entered the inner zone of the open-field more frequently than SD or WKY. SD were more active than WKY in the open-field. WKY took longer to enter the inner zone than SHR or SD. In the elevated-plus maze, SHR spent less time in the closed arms, more time in the open arms and entered the open arms more frequently than SD or WKY. There was no difference between WKY and SD behavior in the elevated-plus maze. SHR released significantly more [3H]norepinephrine in response to glutamate than SD or WKY in both hippocampus and prefrontal cortex while SD prefrontal cortex released more [3H]norepinephrine than WKY. SHR were resilient, cross-fostering did not reduce their ADHD-like behavior or change their neurochemistry. Cross-fostering of SD pups onto SHR or WKY dams increased their exploratory behavior without altering their anxiety-like behavior.
The ADHD-like behavior of SHR and their neurochemistry is genetically determined and not dependent on nurturing by SHR dams. The similarity between WKY and SD supports the continued use of WKY as a control for SHR and suggests that SD may be a useful additional reference strain for SHR. The fact that SD behaved similarly to WKY in the elevated-plus maze argues against the use of WKY as a model for anxiety-like disorders.
PMCID: PMC2711096  PMID: 19549323
19.  Altered Endothelin Receptor Expression and Affinity in Spontaneously Hypertensive Rat Cerebral and Coronary Arteries 
PLoS ONE  2013;8(9):e73761.
Hypertension is associated with arterial hyperreactivity, and endothelin (ET) receptors are involved in vascular pathogenesis. The present study was performed to examine the hypothesis that ET receptors were altered in cerebral and coronary arteries of spontaneously hypertensive rats (SHR).
Methodology/Principal Findings
Cerebral and coronary arteries were removed from SHR. Vascular contraction was recorded using a sensitive myograph system. Real-time PCR and Western blotting were used to quantify mRNA and protein expression of receptors and essential MAPK pathway molecules. The results demonstrated that both ETA and ETB receptor-mediated contractile responses in SHR cerebral arteries were shifted to the left in a nonparallel manner with increased maximum contraction compared with Wistar-Kyoto (WKY) rats. In SHR coronary arteries, the ETA receptor-mediated contraction curve was shifted to the left in parallel with an increased pEC50 compared with the arteries in WKY rats. There was no significant increase in ETB receptor-mediated contraction in SHR coronary arteries. ETA receptor mRNA and protein expression was increased in SHR cerebral arteries compared with the arteries in WKY rats. However, ETA receptor mRNA and protein levels in coronary arteries and ETB receptor protein levels in cerebral and coronary arteries remained unchanged in SHR compared with WKY rats. Meanwhile, phosphorylated ERK1/2 protein was significantly increased in SHR brain and heart vessels.
In SHR cerebral arteries, ETA receptor expression was upregulated. ETA receptor affinity was increased in coronary arteries, and ETB receptor affinity was increased in cerebral arteries. The ERK1/2 activation may be involved in the receptor alterations.
PMCID: PMC3759417  PMID: 24023902
Hypertension  2008;52(2):415-423.
Arterial hypertension is associated with organ dysfunctions, but the mechanisms are uncertain. We hypothesize that enhanced proteolytic activity in the microcirculation of spontaneously hypertensive rats (SHRs) may be a pathophysiological mechanism causing cell membrane receptors cleavage and examine this for two different receptors. Immunohistochemistry of matrix-degrading metalloproteinases (MMP-9) protein shows enhanced levels in SHR microvessels, mast cells, and leukocytes compared to normotensive Wistar-Kyoto (WKY) rats. In-vivo micro-zymography shows cleavage by MMP-1,9 in SHRs that co-localizes with MMP-9 and is blocked by metal chelation. SHR plasma also has enhanced protease activity. We demonstrate with an antibody against the extracellular domain that the insulin receptor-α density is reduced in SHR, in line with elevated blood glucose levels and glycated hemoglobin. There is also cleavage of the binding domain of the leukocyte integrin receptor CD18 in line with previously reported reduced leukocyte adhesion. Blockade of MMPs with broad acting inhibitor (doxycycline, 5.4mg/kg/day) reduces protease activity in plasma and microvessels, blocks the proteolytic cleavage of the insulin receptor, the reduced glucose transport, normalizes blood glucose levels and glycated hemoglobin levels, as well as reduces blood pressure and enhanced microvascular oxidative stress of SHRs. The results suggest that elevated MMP activity leads to proteolytic cleavage of membrane receptors in the SHR, e.g. cleavage of the insulin receptor-binding domain associated with insulin resistance.
PMCID: PMC2677556  PMID: 18606910
Microcirculation; matrix metalloproteinases; insulin receptor; integrin; receptor cleavage; oxygen free radical
21.  Spontaneous white matter damage, cognitive decline and neuroinflammation in middle-aged hypertensive rats: an animal model of early-stage cerebral small vessel disease 
Cerebral small vessel disease (cSVD) is one of the most prevalent neurological disorders. The progressive remodeling of brain microvessels due to arterial hypertension or other vascular risk factors causes subtle, but constant cognitive decline through to manifest dementia and substantially increases the risk for stroke. Preliminary evidence suggests the contribution of the immune system to disease initiation and progression, but a more detailed understanding is impaired by the unavailability of appropriate animal models. Here, we introduce the spontaneously hypertensive rat (SHR) as a model for early onset cSVD and unveiled substantial immune changes in conjunction with brain abnormalities that resemble clinical findings.
In contrast to age-matched normotensive Wistar Kyoto (WKY) rats, male SHR exhibited non-spatial memory deficits. Magnetic resonance imaging showed brain atrophy and a reduction of white matter volumes in SHR. Histological analyses confirmed white matter demyelination and unveiled a circumscribed blood brain barrier dysfunction in conjunction with micro- and macrogliosis in deep cortical regions. Flow cytometry and histological analyses further revealed substantial disparities in cerebral CD45high leukocyte counts and distribution patterns between SHR and WKY. SHR showed lower counts of T cells in the choroid plexus and meningeal spaces as well as decreased interleukin-10 levels in the cerebrospinal fluid. On the other hand, both T and NK cells were significantly augmented in the SHR brain microvasculature.
Our results indicate that SHR share behavioral and neuropathological characteristics with human cSVD patients and further undergird the relevance of immune responses for the initiation and progression of cSVD.
Electronic supplementary material
The online version of this article (doi:10.1186/s40478-014-0169-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4279586  PMID: 25519173
Cerebral small vessel disease; White matter disease; Spontaneously hypertensive rat; Neuroinflammation; T Cells
22.  Acute exercise exacerbates ischemia-induced diastolic rigor in hypertensive myocardium 
SpringerPlus  2012;1:46.
Previous studies have shown that acute exercise preconditions the myocardium from ischemic injury. The purpose of this study was to test whether acute exercise protects the hypertensive myocardium from ischemia-induced diastolic rigor, and to compare the response between normotensive and uncompensated hypertensive hearts. Hearts harvested from female Wistar-Kyoto (WKY; n = 24) and spontaneously hypertensive rats (SHR; n = 27) (age:10–12 weeks) were exposed to ischemia (Langendorff isovolumic preparation; 22 minutes of no flow ischemia and studied following prior conditions of: 1) no exercise (WKY-CON, n=8; SHR-CON, n=8); 2) ischemia initiated one hour post-acute exercise (WKY-1HR, n = 8; SHR-1HR, n = 11); and 3) ischemia initiated 24 hours post-acute exercise (WKY-24HR; n = 8; SHR-24HR, n = 8). Acute exercise consisted of one bout of treadmill running at 25 m/min for 60 minutes. Heart weight was similar between WKY and SHR despite elevated in vivo resting systolic blood pressure and rate pressure product in SHR (P<0.05). During normoxic perfusion, left ventricular (LV) Langendorff performance was similar between WKY and SHR over the post-exercise time course. However, during ischemia, LV diastolic rigor was less in WKY vs. SHR (P<0.05). Acute exercise augmented ischemia-induced LV dysfunction one hour post-exercise in SHR (P<0.05), with gradual recovery by 24 hours post-exercise. These data suggest that acute exercise promotes ischemic diastolic rigor in SHR, even prior to the development of cardiac hypertrophy.
PMCID: PMC3725917  PMID: 23961371
Exercise; Preconditioning; Hypertension; Heart; Ischemia
23.  Cardiovascular Responses Induced by Obstructive Apnea Are Enhanced in Hypertensive Rats Due to Enhanced Chemoreceptor Responsivity 
PLoS ONE  2014;9(1):e86868.
Spontaneously hypertensive rats (SHR), like patients with sleep apnea, have hypertension, increased sympathetic activity, and increased chemoreceptor drive. We investigated the role of carotid chemoreceptors in cardiovascular responses induced by obstructive apnea in awake SHR. A tracheal balloon and vascular cannulas were implanted, and a week later, apneas of 15 s each were induced. The effects of apnea were more pronounced in SHR than in control rats (Wistar Kyoto; WKY). Blood pressure increased by 57±3 mmHg during apnea in SHR and by 28±3 mmHg in WKY (p<0.05, n = 14/13). The respiratory effort increased by 53±6 mmHg in SHR and by 34±5 mmHg in WKY. The heart rate fell by 209±19 bpm in SHR and by 155±16 bpm in WKY. The carotid chemoreceptors were then inactivated by the ligation of the carotid body artery, and apneas were induced two days later. The inactivation of chemoreceptors reduced the responses to apnea and abolished the difference between SHR and controls. The apnea-induced hypertension was 11±4 mmHg in SHR and 8±4 mmHg in WKY. The respiratory effort was 15±2 mmHg in SHR and 15±2 mmHg in WKY. The heart rate fell 63±18 bpm in SHR and 52±14 bpm in WKY. Similarly, when the chemoreceptors were unloaded by the administration of 100% oxygen, the responses to apnea were reduced. In conclusion, arterial chemoreceptors contribute to the responses induced by apnea in both strains, but they are more important in SHR and account for the exaggerated responses of this strain to apnea.
PMCID: PMC3900660  PMID: 24466272
24.  Attenuation of L-Type Ca2+ Channel Expression and Vasomotor Response in the Aorta with Age in Both Wistar-Kyoto and Spontaneously Hypertensive Rats 
PLoS ONE  2014;9(2):e88975.
Age-related vascular diseases are induced by vascular dysfunction, which involves changes in the vasomotor response. The voltage-dependent L-type calcium channel (VDCC) protein is involved in the regulation of vessel function (contraction/relaxation action). In the present study, we evaluated age-related vasomotor function and expression of the signal-related target proteins, including VDCC, using thoracic aorta from both 8- and 40-week old Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). In contraction experiments using aortic rings, vasomotor responses of both phenylephrine-induced contraction and acetylcholine-induced relaxation were significantly attenuated with age in SHR, whereas WKY did not lose activity with age. Contraction induced by angiotensin II was impaired only for the 40-week old SHR among all the rat groups tested, although enhanced AT1R/reduced AT2R expression with age was observed for both WKY and SHR. In contrast, a vasomotor responsiveness to Bay K 8644 (a VDCC agonist) at the initial contraction phase was significantly attenuated in both 40-week WKY and SHR with significant reduction of VDCC protein expression. The reduced VDCC expression in 40-week old rats significantly lowered the relaxation activity of VDCC blockers, such as verapamil and Trp-His, but did not affect that of nifedipine. Taken together, we provided the first evidence that aging caused a reduction of VDCC expression in rat aorta, irrespective of the rat strain, along with diminishment of the therapeutic potential of VDCC blockers.
PMCID: PMC3923070  PMID: 24533163
25.  Impact of Short-Term Treatment with Telmisartan on Cerebral Arterial Remodeling in SHR 
PLoS ONE  2014;9(10):e110766.
Background and Purpose
Chronic hypertension decreases internal diameter of cerebral arteries and arterioles. We recently showed that short-term treatment with the angiotensin II receptor blocker telmisartan restored baseline internal diameter of small cerebral arterioles in spontaneously hypertensive rats (SHR), via reversal of structural remodeling and inhibition of the angiotensin II vasoconstrictor response. As larger arteries also participate in the regulation of cerebral circulation, we evaluated whether similar short-term treatment affects middle cerebral arteries of SHR.
Baseline internal diameters of pressurised middle cerebral arteries from SHR and their respective controls, Wistar Kyoto rats (WKY) and responses to angiotensin II were studied in a small vessel arteriograph. Pressure myogenic curves and passive internal diameters were measured following EDTA deactivation, and elastic modulus from stress-strain relationships.
Active baseline internal diameter was 23% lower in SHR compared to WKY, passive internal diameter (EDTA) 28% lower and elastic modulus unchanged. Pressure myogenic curves were shifted to higher pressure values in SHR. Telmisartan lowered blood pressure but had no effect on baseline internal diameter nor on structural remodeling (passive internal diameter and elastic modulus remained unchanged compared to SHR). Telmisartan shifted the pressure myogenic curve to lower pressure values than SHR.
In the middle cerebral arteries of SHR, short-term treatment with telmisartan had no effect on structural remodeling and did not restore baseline internal diameter, but allowed myogenic tone to adapt towards lower pressure values.
PMCID: PMC4198293  PMID: 25333878

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