Response Gene to Complement 32 Maintains Blood Pressure Homeostasis by Regulating α-Adrenergic Receptor Expression

Abstract

Rationale: Hypertension prevalence is much higher among children and adolescents with low birth weight and greater postnatal weight gain than in individuals with normal birth weight. However, the cause and molecular mechanisms underlying this complication remain largely unknown. Our previous studies have shown that RGC-32 (response gene to complement 32)-deficient (RGC-32^-/-) mice are born significantly smaller but grow faster than their WT (wild type) controls, which allows adult RGC-32^-/- mice to attain body weights similar to those of control mice.

Objective: The objective of this study is to determine whether RGC-32^-/- mice develop hypertension, and if so, to elucidate the underlying mechanisms.

Methods and results: By using a radiotelemetry system, we found that RGC-32^-/- mice exhibit higher mean arterial pressure than WT mice (101±4 versus 119±5 mm Hg), which enabled us to use RGC-32^-/- mice to study the mechanisms underlying low birth weight-related hypertension. The increased blood pressure in RGC-32^-/- mice was associated with increased vascular tone and decreased distensibility of small resistance arteries. The increased vascular tone was because of an increase in the relative contribution of sympathetic versus parasympathetic activity and was linked to increased expression of AT1R (angiotensin II type I receptor) and α1-AdR (α1-adrenergic receptor) in arterial smooth muscles. Mechanistically, RGC-32 regulated AT1R gene transcription by interacting with Sp1 (specificity protein 1) transcription factor and further blocking its binding to the AT1R promoter, leading to suppression of AT1R expression. The attenuation of AT1R leads to reduction in α1-AdR expression, which was critical for the balance of sympathetic versus parasympathetic control of vascular tone. Of importance, downregulation of RGC-32 in arterial smooth muscles was also associated with low birth weight and hypertension in humans.

Conclusions: Our results indicate that RGC-32 is a novel protein factor vital for maintaining blood pressure homeostasis, especially in individuals with low birth weight.

Keywords: angiotensin II; arterial pressure; arteries; blood pressure; hypertension; low birth weight.

Figure 1:. RGC32 deficiency caused hypertension in mice.

A-D, Mean arterial pressure (MBP, A-B) and heart rate (HR, C-D) in 3 month old wild type (WT) and RGC-32 knockout (RGC-32−/−) mice were measured simultaneously and continuously for a 24-hour period. The MBP (B) and HR (D) were calculated by averaging the 24 hour recording for all mice in each group. Shown are the means ± SEM. *P<0.05 vs. WT, n=6. E, Whole-mount double-staining of lectin (green-ECs) and α-SMA (red-VSMCs) in retinal vessels of WT and RGC-32 mice. Arrows indicate veins; arrowheads indicate α-SMA-positive arteries. Scale bar=50μm. F, The ratios of artery to vein diameters (A:V) were calculated by averaging the diameters of all retinal arteries and veins in the same microscope fields. *P<0.05 compared to the WT, n=5.

Figure 2:. RGC-32 deficiency decreased arterial distensibility and increased arterial vascular tone.

A-C, RGC-32 deficiency (RGC32−/−) reduced the distension of mesenteric arteries (MRA) in response to intraluminal pressure in Ca²⁺-free and EGTA-containing PSS passive condition as measured by myograph. A, Vessel images with or without 70 mmHg intraluminal pressure; Scale bar=100 μm. B-C, Quantification of the vessel lumen diameters and artery wall to lumen ratios. *P<0.05 vs wild type (WT) vessels (n=6). D, RGC32−/− MRA showed greater resistance to intraluminal pressure than the WT vessels.*P<0.05 vs. WT vessels (n=5). E, Distensibility (D20) of the MRA from 3-month-old WT and RGC32−/− mice over a range of intraluminal pressures as indicated was measured by cultured myograph system and calculated as described in Methods. *P<0.05 vs. WT MRA (n=6). F, Vessel tone of MRA at the given pressures was calculated as: [1-(active diameter/passive diameter)]*100. *P<0.05 vs. WT vessel (n=6). G-H, Dose-dependent contractile response of mesenteric arteries (MRA) to KCl or norepinephrine (NE) at 70 mmHg intraluminal pressure. *P<0.01 vs. WT MRA at the same dosage of the stimuli (n=6). KCl or NE caused greater contraction responses in RGC-32−/− MRA compared to the WT vessels.

Figure 3:. RGC32 deficiency altered the balance of sympathetic vs. parasympathetic influences on cardiovascular function.

A-B,RGC32 deficiency (RGC32−/−) suppressed the tachycardic response to atropine (Atrop), indicating a decreased parasympathetic tone in 3 month old RGC32−/− mice as compared to the wild type (WT) mice. C-D, RGC32−/− increased the bradycardic response to propranolol (Prop), suggesting an increased sympathetic tone in RGC32−/− mice. Changes in heart rates in A-D were obtained by subtracting the resting heart rates measured prior to each treatment. E-F, The resting systolic blood pressure (SBP) was 117±6 mmHg in WT (n=6) and 144±10 mmHg in RGC32−/− mice (n=6). RGC32−/− mice exhibited a much greater reduction in SBP with a ganglion blockade by hexamethonium (Hexa) than WT mice, suggesting that RGC32−/− increased the neurogenic sympathetic control of vascular resistance. G-H, The resting SBP was 121±5 mmHg in WT (n=6) and 146±9 mmHg RGC32−/− mice (n=6). RGC32−/− mice exhibited a much greater reduction in SBP with the α1-adrenergic blockade by prazosin (Praz) compared to the WT mice, further demonstrating that RGC32−/− increased the neurogenic sympathetic control of vascular resistance. The changes of SBP were obtained by subtracting the resting SBP measured prior to each treatment. *P<0.05 compared to the corresponding WT mice in each treatment (unpaired t-test, n=6).

Figure 4:. RGC-32 deficiency increased the expression of α-adrenergic receptor in small/resistance arteries.

A, RGC-32 deficiency (RGC-32−/−, 3 month old) did not alter plasma epinephrine (E) and norepinephrine (NE) levels. B, The mRNA expression of different α-adrenergic receptors (α1A, α1B, α1D, α2, A-C) in mesenteric arteries (MRA) was measured by qPCR. *P<0.05 vs wild type (WT) mouse MRA (n=6). C, RGC-32−/− increased α1-adrenergic receptor (α1-AdR) protein expression in MRA as measured by Western blot and quantified by normalizing to the α-Tubulin level. *P<0.05 vs WT (n=6). E, Time course systolic blood pressure (SBP) changes following phenylephrine (PE, 1mg/kg, i.p.) stimulation as determined by a radiotelemetry system. *P<0.05 vs. WT mice (n=6). F, Blood pressure elevation in RGC32−/− mice in response to PE was significantly greater than in WT mice. *P<0.05 vs. WT mice (n=6). G, Osmotic pump administration of NE (4.2 mg.day⁻¹ kg⁻¹) for 14 days caused a more blood pressure increase in RGC32−/− mice than in WT mice, as measured by noninvasive CODA blood pressure monitor system, *P<0.05 vs. NE-treated WT mice (n=6).

Figure 5:. RGC32 deficiency increased blood pressure through angiotensin II (Ang II) receptor (AT1R)-mediated upregulation of α-adrenergic receptor.

A-B, Compared to WT, RGC-32 deficiency (RGC-32−/−) exhibited a much greater angiotensin II (Ang II, 1 ug/kg, i.p)-induced increase of systolic blood pressure (SBP) (A) or Azilsartan (1 ug/kg, i.p)-induced decrease of SBP (B). The SBP was measured by radiotelemetry. *P<0.05 vs WT mice (n=6). C, SBP increase in response to Ang-II (0.75mg.kg⁻¹ day⁻¹) stimulation by minipump infusion for 14 days. *P<0.05 vs. Ang-II-treated WT mice, n=6. D, RGC-32 deficiency increased ATR mRNA expression in mesenteric arteries (MRA) as measured by qRT-PCR and normalized to the cyclophilin level. *P<0.05 vs WT MRA for each receptor (n=5). E, RGC-32 deficiency increased ATR1 protein expression in MRA as measured by Western blot and normalized to α-tubulin level. *P<0.05 vs. WT MRA (n=5). F, Forced expression of AT1R rescued RGC-32-suppressed α-adrenergic receptor (α1A-AdR) expression in primary cultured rat VSMCs. Adenoviral vectors expressing GFP (Ad-GFP), RGC-32 (Ad-RGC32), or ATR1 (Ad-ATR1) were transduced individually or in combination into vehicle (−) or Ang II-treated VSMCs as indicated. α1-AdR protein expression was assessed by Western blot. G, α1-AdR protein expression shown in E was normalized to α-Tubulin. *^,&P<0.05 vs. Ad-GFP group; ^#P<0.05 vs. Ad-RGC32 group; ^$P<0.05 vs. Ad-GFP+Ang II group; ^@P<0.05 vs. Ad-RGC32+Ang II group (n=3).

Figure 6:. RGC-32 inhibited AT1R gene transcription by interacting with Sp1 and thus blocking its binding to ATR1 promoter.

A, RGC-32 inhibited Angiotensin II (Ang II)-induced AT1R promoter activity. Primary rat VSMCs were transduced with adenovirus expressing GFP (AdGFP) or RGC-32 (AdRGC32) and transfected with AT1R promoter reporter construct for 48 h followed by vehicle or Ang II treatment (200 nM) for 24 h. Luciferase assay was performed. *P<0.01 compared with AdGFP group with vehicle treatment. ^#P<0.01 compared with AdGFP group with Ang II treatment (n=3). B, RGC32 inhibited Sp1-mediated AT1R promoter activity. Primary rat VSMCs were transduced with AdGFP or AdRGC32 and cotransfected with AT1R promoter reporter construct and control (−) or Sp1 plasmid as indicated for 48 h followed by vehicle or Ang II treatment for 24 h. Luciferase assay was performed. *P<0.01 compared with AdGFP alone group with vehicle treatment. ^#P<0.01 compared with Sp1/AdGFP group with vehicle treatment. ^@P<0.01 compared with AdGFP group with Ang II treatment. ^&P<0.01 compared with Sp1/AdGFP group with Ang II treatment (n=3). C-F, Co-IP with endogenous proteins indicated that RGC32 physically interacted with Sp1. Rat VSMCs were treated with vehicle (−) or Ang II (+, 200 nM) for 24 h. Cell lysates were immunoprecipitated with normal IgG, Sp1 (C), or RGC32 (E) antibody. The immunoprecipitates were immunoblotted (IB) with RGC32 and Sp1 antibodies (C & E). RGC32 pulled down by Sp1 (D) or Sp1 pulled down by RGC32 (F) as shown in C and E was quantified by normalizing to the Sp1 level in the input. *P<0.01 compared with IgG pulldown group. ^#P<0.01 compared with Sp1- (D) and RGC32- (F) pulldown group with vehicle treatment (n=3). The interaction between RGC32 and Sp1 was attenuated by Ang II induction. G-H, RGC32 suppressed Ang II-enhanced Sp1 binding to the AT1R promoter in a chromatin setting. Primary rat VSMCs were transduced with AdGFP or AdRGC32 followed by vehicle or Ang II treatment for 24 h. ChIP assays were performed, and the Sp1 binding to the GC box in AT1R promoter was detected by semi-quantitative PCR (G) and qPCR (H). *P<0.01 compared with vehicle-treated group; ^#P<0.01 compared with Ad-GFP group treated with Ang II (n=3).

Figure 7:. RGC-32 was down-regulated in arteries of human low birth weight individuals and hypertensive patients.

A, The umbilical arteries of human individuals with normal (NBW) or low birth weight (LBW) were immunostained with α-SMA (red) and RGC-32 (green) antibodies or normal IgG (negative control). DAPI stains nuclei. B-C, RGC32 expression in the umbilical arteries were examined by Western blot and quantified by normalization to α-Tubulin. *P<0.05 vs. NBW, n=3. D-E, RGC-32 expression in thoracic aorta of healthy individuals (Normotensive, NT) and hypertensive patients (Hypertensive, HT) was detected by immunohistochemistry staining using RGC-32 antibody. The RGC-32-positive cells were counted and quantified relative to total VSMC numbers in the artery media. *P<0.01 compared to NT, n=5.