Male normotensive Wistar-Kyoto rats (WKY) and Spontaneously Hypertensive rats (SHR) were obtained from Charles River (Maastricht, the Netherlands). All animals were housed separately and had free access to standard food (SRMA-1210; Hope Farms, Woerden, the Netherlands) and tap water. All experiments were conducted according to institutional guidelines. This investigation conforms to the Guide for Care and Use of Laboratory Animals as published by the US National Institutes of Health (NIH Publication No.85-23, revised 1996), and all experimental protocols were approved by the Animal Ethics Committee of the Maastricht University.

Small mesenteric arteries (MA) of 6 week old WKY (n =27) and SHR (n =27) were exposed to altered blood flow in vivo as described previously [6, 14, 17, 31]. Briefly, rats were anesthetized with isoflurane (Abbott, Kent, UK) and after laparotomy, local blood flow was reduced (LF) by distal ligation of three alternate second order MA branches. The MA between these had compensatory enhanced flow (HF). We previously observed in WKY that the blood flow averages 10% and 200% in LF and HF respectively when compared to second order MA outside of the surgical area (normal flow, NF) and that these interventions ultimately result in inward hypotrophic (LF) and outward hypertrophic arterial remodeling (HF), respectively [6, 14, 31]. Animals received buprenorphine (0.05 mg/kg, s.c.; Schering-Plough, Utrecht, Netherlands) as an analgesic at three time-points: before surgery, at the end of the day of surgery and the next morning.

At 24 hours after flow-modifying surgery, NF, HF and LF MA segments were isolated and pooled for each individual WKY and SHR rat (n = 9). To isolate RNA, Trizol reagent (Invitrogen, Leek, NL) in combination with RNeasy Mini Kit (Qiagen, Venlo, NL) was used. RNA (100 ng) was then used for first-strand cDNA synthesis with Ready-To-Go You-Prime First-Strand Beads (GE healthcare, Diegem, BE) and pd(N)₆ random hexamer primer (GE healthcare, Diegem, BE). Plasmids containing gene fragments of rat MMP2, TSP1 and cyclophilin were used as standard curves, while a pool of cDNA of all samples was used for standard curves for the eNOS, sGCα1 and PKG1β genes. Taqman RT-PCR for MMP2, TSP1 and cyclophilin was performed in an ABI Prism 7700 SDS cycler (Applied-biosystems, Bleiswijk, NL) in combination with the appropriate fluorogenic probes, primer and qPCR MasterMix (Eurogentec, Maastricht, NL). Taqman RT-PCR for eNOS, sGCα1, PKG1β and cyclophilin was performed in a MyiQ iCycler (Biorad, Zwijndrecht, NL) with primers and qPCR MasterMix containing the DNA intercalating dye, SYBR-green. Each PCR reaction was performed in duplicate wells using the following conditions, 10 min at 95°C, followed by a total of 40 cycles of 15 s at 95°C and 1 min at 60°C (15 s at 95°C and 30 s at 58°C for SYBR-green). Gene expression levels were normalized to the house-keeping gene cyclophilin in each sample.

To evaluate consequences of altered gene expression for vasomotor control, 2 mm arterial segments were harvested at 24 (n=4) and at 32 hours (n=8) after flow modifying surgery in 6 week old WKY and SHR. Preparations were mounted in a 5 ml organ chamber (Danish Myotechnology, Aarhus, DK) filled with Krebs Ringer bicarbonate solution (KRB; 37°C; 95% O₂ / 5% CO₂) between an isometric force transducer and a displacement device. Their diameter was set at 90% of that of the resting vessel at a transmural pressure of 100 mmHg. Effects of the endothelium-dependent vasodilator acetylcholine, the NO-donor Na-nitroprusside and of the non-selective NOS-inhibitor N^ω-L-nitro arginine methyl ester (L-NAME) were evaluated in the presence of 3 μmol/L indomethacin during contractions induced by an equiosmotic KRB solution containing 40 mmol/L K⁺. These steps were taken to rule out influences of prostaglandins and endothelium-derived hyperpolarizing factors. Because depolarizing high K⁺ buffer can stimulate release of neurotransmitters from peri-arterial nerves, experiments were initiated after exposure of the arteries to phenoxybenzamine (1 μmol/L, 10 min) and capsaicin (1 μmol/L, 20 min) which irreversibly block α-adrenoceptors and peri-arterial sensorimotor nerves, respectively [32].

Protein levels were investigated in view of discrepancies between mRNA levels on the one hand and vasomotor responses on the other hand. At 32 hours and at 40 hours after flow-modifying surgery, NF, HF and LF MA segments (n=9, each group) were isolated and pooled from 3 WKY and 3 SHR rats and snap-frozen in liquid nitrogen. Pooled arteries were crunched at -80°C and resuspended in ice cold RIPA buffer (50 mM HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% Na-deoxycholate and 1 mM EDTA) containing protease and phosphatase-inhibitors (1mM PMSF, 1 mM NaVO₄, 1 mM NaF, 1 µg/ml aprotinin, 1 µg/ml pepstatin and 1 µg/ml leupeptin) to extract total protein. The lysates were cleared by centrifugation. Protein content of the lysates was determined with the BCA method [33]. Lysates were subsequently run on a criterion bis/tris gradient gel, and blotted to a nitrocellulose membrane. Membranes were blocked with 1:1 oddysey blocking buffer:PBS and were subsequently incubated with antibodies diluted in 1:1 oddysey blocking buffer:PBS against: eNOS (BD Transduction Laboratories, Franklin Lakes, USA, 1:2500), sGCα1 (Santa Cruz, Santa Cruz, USA 1:2000), PKG1β (Biovision, Zurich, CH, 1:500), MMP2 (Santa Cruz, Santa Cruz, USA 1:500,detecting the pro (72kDa)- and the active (62kDa) form of MMP2) and TSP-1 (Abcam, Cambridge, USA, 1:500). Primary antibodies were detected with secondary antibodies labeled with the near infrared dye IRdye800 and visualized with an odyssey detection system. TSP-1 antibody was detected with secondary antibodies labeled with biotin. Biotin was subsequently detected with IRdye800 labeled streptavidin. Protein expression levels were normalized to the house-keeping protein GAPDH in each run.

To investigate whether hepI could reverse inward arterial remodeling in hypertension, we used MA segments of 12 week old SHR in organ culture as described by Bakker et al. [10, 34]. The vessels were mounted on two glass micropipettes in a stainless steel organ chamber. Vessels were pressurized at 80 mmHg (no flow) and incubated in Hanks balanced salt solution (HBSS) for 60 min at 37°C, before baseline measurements of diameter and reactivity were obtained. Thereafter the vessels were exposed intra-luminally as well as extra-luminally to culture medium (Leibovitz (n=6) or 5% dFCS-DMEM (n=6)) with or without 1 μmol/L hepI for 3 days. The parallel experiments with Leibovitz and 5% dFCS in DMEM yielded quantitatively identical results. The data were pooled for subsequent analysis.

At the start of the experiments and at daily intervals, passive pressure-diameter (P/D) curves were constructed [13]. The culture medium was replaced by Ca²⁺-free HBSS containing 10 μmol/L Na-nitroprusside to inactivate arterial smooth muscle tone. The cannulated arterial segments were pressurized using a feedback controlled pressure source (Living Systems Instrumentation, Burlington, USA) and placed on the stage of an invertoscope (Nikon TMS, Abingdon, USA) equipped with a video camera (Stemmer) and a digital device (LSI) for recording of lumen diameter. Intra-arterial pressure was increased in 10 mmHg steps from 20 to 120 mmHg. After each step, the vessel was allowed to equilibrate until a stable diameter was reached (2 minutes). Findings were expressed as percentage change from vessel diameter on day 0 at 80 mmHg. From the P/D relationships arterial distensibility was calculated by the following formula: DC = (1/ΔP)*(ΔD/D_n-1)*100 (DC = distensibility, P = intraluminal pressure, D = diameter at a given intraluminal pressure). After the highest pressure was reached, pressure was returned to 80 mmHg and the HBSS-solution was replaced by culture medium with or without 1.0 μmol/L hepI.

On day 0 and day 3 of the culture period the viability of the vessels was verified by recording diameter changes at 80 mmHg in response to 1 μmol/L serotonin, 10 μmol/L phenylephrine and 1 μmol/L acetylcholine in HBSS. Finally the vessels were fixed in neutral buffered formalin at 80 mmHg and 37°C and stored in 70% ethanol.

Fixed vessels were embedded in paraffin and cross-sections (4 μm) were stained with Lawson’s solution (Boom, Meppel, the Netherlands) to visualize the internal and external elastic laminae. Cross-sectional area (mCSA), thickness, cellular density of the tunica media, and the lumen diameter were determined as previously described [6, 13, 14, 17, 26, 31].

Leibovitz medium (Invitrogen, Leek, NL) was supplemented with 1 mg/mL albumine (Sigma Aldrich, Zwijndrecht, NL), 20 mg/mL ciprofloxacin (Bayer, Leverkusen, DE), 100 U/mL penicillin, 100 μg/mL streptomycin and 2.5 U/mL amphotericin B (Invitrogen, Leek, NL). DMEM (Invitrogen, Leek, NL) was supplemented with 2.5 mM glutamine, 100 IU/mL penicillin, 100 μg/mL streptomycin and 5% dialyzed fetal-calf serum. All pharmacological agents were purchased from Sigma Aldrich, (Zwijndrecht, NL). The hepI peptide (ELTGAARKGSGRRLVKGPD) was synthesized by Anaspec Inc (San Jose, USA) and purified by HPLC [21]. The peptide was confirmed to be >95% pure by HPLC and mass spectrometry.

Results are expressed as means ± SEM. Statistical significance of differences between groups was evaluated by analysis of variance (ANOVA for consecutive measurements for pressure-diameter curves) or 1-way ANOVA followed by Bonferroni or paired t-test (SPSS 11.5). A value of P<0.05 was considered significant.

Fig. (1) illustrates expression of genes encoding protein kinase G (PKG) and candidate modulators and targets of this enzyme in mesenteric small arteries of 6 week old WKY and SHR rats. In control arteries with normal flow (NF), mRNA levels were high for sGCα1 and PKG1β, intermediate for eNOS, and low for MMP2 and TSP1. Although expression of eNOS tended to be smaller in NF of SHR, the expression levels of all five genes did not differ significantly between NF arteries of 6 weeks old SHR and WKY rats.

Fig. (1). mRNA expressions of eNOS, sGCα1, PKG1β, TSP1 and MMP2 in mesenteric small arteries exposed to normal blood flow (NF, black bars) or for 24 hours to increased blood flow (HF, hatched bars) or reduced blood flow (LF, crosshatched bars) in 6 week old Wistar Kyoto (WKY) rats and Spontaneously Hypertensive rats (SHR) in vivo, normalized to the housekeeping gene cyclophylin. Results are expressed as the mean ± SEM (n = 6). * p<0.05 versus NF.

Fig. (2). In vitro contractile reactivity of mesenteric small arteries of 6 week old WKY rats (panel A) and SHR rats (panel B) exposed to normal flow (black bars) or for 32 hours to increased (hatched bars) or reduced blood flow (cross-hatched bars). Means ± SEM (n = 8-11). Normalized diameter and maximal contraction in response to 125 mmol/L K+ (top left) and the effect of indomethacin and L-NAME on contractile responses to 40 mmol/L K+ (top right) are shown along with the relaxing responses to acetylcholine during K+-induced contraction in the presence of indomethacin (bottom left) and the relaxing responses to Na-nitroprusside during K+-induced contraction in the presence of indomethacin and L-NAME (bottom right) for NF (closed circle), HF (open circle) and LF (closed triangle). *p<0.05 versus NF; #p<0.05 INDO versus INDO + L-NAME.

Fig. (3). Changes in protein expression of eNOS (A), sGCα1 (B), PKG1β (C), MMP2 (pro/act, D/E) and TSP1 (F), in mesenteric small arteries exposed for 32 and 40 hours to normal (black bars), increased (hatched bars) or reduced (crosshatched bars) blood flow in 6 week old WKY and SHR rats in vivo. Means ± SEM (n = 6). *p<0.05 versus NF.

Fig. (4). Effects of time (control, top left) and 1 µmol/L hepI (hepI, top right) on passive pressure-diameter relationships in mesenteric small arteries (MA) of 12 week old SHR during organ culture. Calculated distensibility for control MA (day 0 and day 3, bottom left) and hepI treated MA (day 0 and day 3, bottom right). Values are shown as means ± SEM (n = 12). Closed circle, day 0; open circle, day 1; closed triangle, day 2; open triangle, day 3. *p<0.05, versus day 0.

Changes in gene expression in MA exposed for 24 hours to altered blood flow in vivo are summarized in Fig. (1). Consistent with earlier RNA microarray observations [17, 18], the expression of sGCα1 was significantly reduced and that of TSP1 was markedly increased in arteries from WKY rats exposed to high blood flow (HF). Surprisingly, the findings were very similar in vessels exposed to increased (HF) or reduced (LF) blood flow. In both HF and LF arteries of WKY rats, the expression of eNOS, sGCα1 and PKG1β was significantly reduced, whereas TSP1 was markedly increased and MMP2 was not appreciably altered. In HF of SHR, qualitatively and quantitatively similar findings were obtained, except that there was a less marked downregulation of eNOS possibly due to the already reduced basal levels. Also in arteries of SHR exposed to reduced blood flow (LF), largely similar results were obtained, with the exception that eNOS and TSP1 expression were affected to a lesser extent. Collectively these results indicate that either an increase or a decrease in blood flow both lead to a concurrent reduction of eNOS, sGCα1 and PKG1β and to a marked increase of TSP1 mRNA levels within 24 hours in rat mesenteric small muscular arteries.

To evaluate potential functional consequences of the observed downregulation of the NO/sGC/PKG pathway, we recorded arterial contractile reactivity in vitro in MA of 6 week old WKY and SHR rats. At 24 hours after flow-modifying surgery there were no statistically significant differences between NF, HF or LF arteries (n = 4-6, data not shown). Because changes in the abundance and activity of proteins lag behind changes in mRNA expression, functional analyses were repeated at 32 hours after altered blood flow in vivo. These results are summarized in Fig. (2). The diameter of WKY LF arteries was significantly reduced which is consistent with the rapid inward remodeling of LF in vivo [14]. L-NAME significantly increased the contractile response to 40 mmol/L K⁺ in NF arteries of WKY and SHR. Relaxing responses of depolarized vessels to acetylcholine (in the presence of indomethacin) and to Na-nitroprusside (in the presence of indomethacin and L-NAME) did not differ between NF, HF or LF arteries (Fig. 2A). These findings indicate that the marked and concurrent reductions of the mRNA levels for eNOS, sGCα1 and PKG1β were not accompanied by reduced relaxing responses to basal and stimulated endothelium-derived or exogenous NO during initiation of outward (HF) or inward (LF) flow-related arterial remodeling in WKY. Similar results were obtained in SHR, as shown in Fig. (2B). In contrast to WKY, the diameter of SHR LF arteries did not differ from that of NF and HF arteries.

Fig. (3) summarizes changes in protein expression in MA exposed for 32 (left panel) and 40 hours (right panel) to altered blood flow in vivo. These time-points were chosen to allow protein synthesis to occur and are based on the 24 hour gene expression experiments and the earlier observation that in low flow conditions specifically, the inward remodeling response manifests within 2 days [14]. In both HF and LF arteries of WKY and SHR rats, the protein levels of eNOS, sGCα1, PKG1β and MMP2 (pro/act) were not significantly altered after 32 and 40 hours of flow modifying surgery (Fig. 3A-E). Protein expression for TSP1 was significantly increased in LF arteries of both WKY and SHR (Fig. 3F). After 40 hours of exposure to LF, protein expression of TSP1 was below threshold in NF, HF and LF arteries.

To evaluate structural consequences of upregulated TSP1 mRNA and protein expression, isolated arteries were exposed to hepI under constant conditions of pressure (80 mmHg) and flow (no flow) in vitro. These experiments were deliberately performed in arteries of 12 week old SHR that display an inward eutrophic remodeling which contributes to the increased peripheral resistance and blood pressure [24, 29]. In the untreated arteries, the diameter at 80 mmHg and the relationship between passive diameter and transmural pressure did not change during the 3 day culture period (Fig. 4). The presence of 1 μmol/L hepI, however, resulted in an increase of the diameter at 80 mmHg and in an upward shift of the pressure-diameter curves (p < 0.05 for day 1, day 2 and day 3)(Fig. 4). Arterial structural diameter was increased by 7.5 ± 1.9 % within 24 hours and increased further to +12.6 ± 2.4 % after 3 days (Fig. 4). Exposure to hepI did not modify calculated distensibility (Fig. 4), general arterial histology (not shown), and did not result in statistically significant changes in media cross sectional area (13693 ± 4043 μm² versus 12881 ± 3182 μm²) or cell number (52 ± 11 versus 54 ± 8 nuclear profiles/section) indicating that the hepI-induced outward remodeling was eutrophic in nature.

In an additional set of experiments we tested whether hepI displayed vasomotor effects. Contractile responses of freshly isolated arteries to 25 mmol/L K⁺ were reduced by acetylcholine and enhanced by indomethacin and L-NAME indicating intact endothelium. Depolarization-induced contractions were not modified by 0.0001 - 1.0 μmol/L hepI indicating that hepI does not display major acute vasomotor effects. After 3 days in vitro, 1 μmol/L serotonin and 10 μmol/L phenylephrine reduced arterial diameter to 45 ± 4% and 47 ± 3% of baseline, respectively, in untreated vessels and to 44 ± 3% and 46 ± 4% of baseline, respectively, in arteries that had been exposed to hepI. Moreover, 1 μmol/L acetylcholine reversed the serotonin-induced constriction by 66 ± 5% in untreated vessels and by 63 ± 5% in hepI treated arteries. These findings indicate that exposure to hepI for three days did not adversely affect vasoconstriction or endothelium-dependent vasodilatation.