Altered reactivity of resistance vasculature contributes to hypertension in elastin insufficiency

Abstract

Elastin (Eln) insufficiency in mice and humans is associated with hypertension and altered structure and mechanical properties of large arteries. However, it is not known to what extent functional or structural changes in resistance arteries contribute to the elevated blood pressure that is characteristic of Eln insufficiency. Here, we investigated how Eln insufficiency affects the structure and function of the resistance vasculature. A functional profile of resistance vasculature in Eln^+/− mice was generated by assessing small mesenteric artery (MA) contractile and vasodilatory responses to vasoactive agents. We found that Eln haploinsufficiency had a modest effect on phenylephrine-induced vasoconstriction, whereas ANG II-evoked vasoconstriction was markedly increased. Blockade of ANG II type 2 receptors with PD-123319 or modulation of Rho kinase activity with the inhibitor Y-27632 attenuated the augmented vasoconstriction, whereas acute Y-27632 administration normalized blood pressure in Eln^+/− mice. Sodium nitroprusside- and isoproterenol-induced vasodilatation were normal, whereas ACh-induced vasodilatation was severely impaired in Eln^+/− MAs. Histologically, the number of smooth muscle layers did not change in Eln^+/− MAs; however, an additional discontinuous layer of Eln appeared between the smooth muscle layers that was absent in wild-type arteries. We conclude that high blood pressure arising from Eln insufficiency is due partly to permanent changes in vascular tone as a result of increased sensitivity of the resistance vasculature to circulating ANG II and to impaired vasodilatory mechanisms arising from endothelial dysfunction characterized by impaired endothelium-dependent vasodilatation. Eln insufficiency causes augmented ANG II-induced vasoconstriction in part through a novel mechanism that facilitates contraction evoked by ANG II type 2 receptors and altered G protein signaling.

vascular elasticity is an essential property of the closed circulatory system, where blood vessels serve two key functions. In large conducting vessels, elasticity enables vessels to store energy during systole and release it in diastole, thereby allowing the heart to function at an optimal rate and stroke volume. In resistance arteries, elasticity is important for maintaining tone and for regulating blood pressure. The structural component of the vessel wall that imparts elasticity is the extracellular matrix protein elastin (Eln). In the elastic conducting arteries, Eln is organized into interconnected concentric sheets, or lamellae, that separate the smooth muscle cell layers (10). The relative amount of Eln decreases as pulse pressure decreases down the vascular tree to where, in the muscular resistance arteries, elastic layers are found predominantly at the inner (internal elastic lamina) and sometimes outer (external elastic lamina) borders of the smooth muscle-containing medial compartment.

Loss-of-function mutations in Eln lead to supravalvular aortic stenosis [SVAS; Online Mendelian Inheritance in Man (OMIM) database no. 185500], an autosomal dominant disease that predominantly affects large elastic vessels in the vascular system (11, 34, 49). SVAS is also a component of Williams-Beuren syndrome (WBS; OMIM database no. 194050), which results from a microdeletion of a region of human chromosome 7 that includes ELN. These mutations result in ELN haploinsufficiency, and it is the decreased amount of normal Eln that is responsible for disease pathogenesis. Vascular features in SVAS and WBS include narrowing of large conducting vessels, such as the aorta and pulmonary artery, increased vascular stiffness, and hypertension (45). Eln insufficiency also can arise from growth inhibition during the developmental period when Eln deposition is rapid, leading to a predisposition to hypertension later in life (8).

Previously, we showed that Eln heterozygous (Eln^+/−) mice develop structural and functional cardiovascular changes that have many similarities to humans with SVAS (15, 31, 33). The hypertensive phenotype of the Eln^+/− mouse is associated with marked alterations in the mechanical properties of large conduit vessels. It is not clear, however, whether these large vessel changes are sufficient to account for the persistent hypertension in these animals since peripheral resistance, and hence blood pressure, is primarily determined by small resistance arteries. Therefore, we tested the hypothesis that alterations in the structural and functional properties of the resistance vasculature contribute to the high blood pressure in Eln insufficiency. To determine the effect of Eln insufficiency on the peripheral vasculature, we evaluated the morphology and reactivity of small resistance arteries from the mesenteric vascular bed of Eln^+/− mice. Our results show altered reactivity of resistance arteries in Eln haploinsufficient mice characterized by endothelial dysfunction and altered G protein signaling by ANG II receptors. These findings have implications for understanding and treating elevated blood pressure in individuals with SVAS and in other conditions where Eln function is compromised.

MATERIALS AND METHODS

Animals.

All experiments were performed in accordance with protocols approved by the Animal Studies Committee of Washington University. All mice were provided access to food and water ad libitum and housed in our institution's animal facility maintained at a constant temperature of 22°C and a 12:12-h light-dark cycle. All experiments were performed using 3- to 6-mo-old male mice with a C57BL/6 mouse genetic background. The generation and backcrossing of Eln^+/− mice have been previously described (15, 32). Whenever possible, littermates from Eln^+/− crosses were used for all experiments.

Histological analysis of wild-type and Eln^+/− aortae and mesenteric arteries.

Isolated thoracic aortae and mesenteric arteries (MAs) from wild-type (WT) and Eln^+/− mice were cleaned of adipose tissue at 4°C and fixed in 10% formalin fixative. After being embedded in paraffin, 10-μm cross-sections of vessels were processed for hematoxylin and eosin or Verhoeff-van Gieson staining using the services of the Washington University's Department of Pathology core facility.

Vessel isolation and preparation.

Previously described methods were followed for vessel isolation and preparation (12, 42). Briefly, male mice were anesthetized with ketamine (87 mg/kg ip) and xylazine (13 mg/kg ip). The small intestine was accessed after the abdomen was shaved, and a midline incision was made first through the skin and then through the abdominal muscle. The entire intestine was excised and placed in chilled 1% albumin in physiological salt solution (MOPS, pH 7.4) containing (in mM) 144 NaCl, 3.0 KCl, 2.5 CaCl₂, 1.5 MgSO₄, 2.0 pyruvate, 5.0 glucose, 0.02 EDTA, and 1.21 NaH₂PO₄. The mouse was then euthanized by cervical dislocation. Using a pair of fine forceps and with the aid of a dissecting microscope, adipose tissue was carefully removed from second-order MAs. Sections of isolated vessels 2–3 mm in length were excised and immediately placed in an organ chamber for mounting on glass pipettes.

Vessel superfusion and diameter measurements.

An excised piece of the MA was transferred into an organ bath (2.5 ml volume) containing MOPS and mounted on the stage of an inverted microscope (Zeiss Axiovert S100TV). The vessel was cannulated on one end with glass perfusion pipette and occluded on the other end with a collecting pipette such that no luminal flow was allowed during the experiment. The lumen of the vessel was then filled with 1% albumin in MOPS buffer. The vessel was observed with a video camera system (MTI CCD-72) to track and record changes in vessel diameter in response to vasoactive agents. The baseline internal diameter of the vessel was measured online at 60 mmHg and 37°C with a computerized diameter tracking system (sampling rate: 10 Hz, Diamtrak 3 Plus, Montech). The vessel diameter in the presence of various vasoactive agents was measured after at least 30 min of equilibration, when the bath solution was replaced with warmed MOPS buffer containing freshly prepared solution.

Vessel reactivity assays.

Excised pieces of small MAs were transferred and cannulated under no-flow conditions in an organ bath as described previously (12). After vessels were equilibrated for 30 min, the bath solution was replaced with warmed MOPS containing increasing concentrations of either phenylephrine (PE), ANG II, or the ANG II type 2 receptor (AT₂R) agonist novokinin, and changes in vessel diameter was continuously recorded. Before each experiment began, endothelial integrity was evaluated by a constriction with 5 μM PE followed by vasodilation with 1 μM ACh in the presence of 5 μM PE. Vessels were judged to be suitable for experiments if they showed at least 50% relaxation to PE. For Eln^+/− vessels, the vasodilation criterion was lowered to 10% after we observed in preliminary experiments that these vessels responded poorly to ACh. For vasodilatation experiments, vessels were constricted with 5 μM PE after 30 min of equilibration at 37°C. After constriction had reached steady state (∼1 min), the bath solution was changed to MOPS containing 5 μM PE and ACh or the endothelium-independent vasodilators sodium nitroprusside (SNP) or isoptroterenol (ISO) at various concentrations (10⁻⁹–10⁻⁴ M). After the highest ACh, SNP, or ISO concentration was applied, the bath solution was replaced with MOPS to allow complete relaxation. For experiments in which ROS were scavenged, Rho kinase was inhibited, or AT₂Rs were blocked, freshly prepared cell-permeable superoxide dismutase mimetic Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP; 100 μM), the Rho kinase inhibitor Y-27632 (0.1, 1.0, or 5 μM), or the AT₂R antagonist PD-123319 (10 μM) was applied to the vessel bath continuously for 20 min followed by the application of increasing concentrations of ANG II in the presence of the inhibitor or antagonist.

Ex vivo cytosolic Ca²⁺ measurements.

G protein-coupled receptor (GPCR) agonist-elicited rise in cytosolic Ca²⁺ in pressurized small MAs was measured using the Ca²⁺-sensitive dye fura-2 in accordance with our previously described methods (12). Briefly, cannulated WT and Eln^+/− second- or third-order MAs were extraluminally loaded with fura-2 AM solution (final concentration of 5 μM in MOPS buffer containing 0.02% pluronic acid) for 15 min after 20 min of vessel equilibration at 37°C in MOPS buffer. After being loaded with dye, vessels were allowed to equilibrate for 20 min. Fluorescent emissions during excitation at 340 nm (F₃₄₀) and 380 nm (F₃₈₀) were simultaneously recorded using a photon multiplier-based detection system (sampling rate: 10 Hz, C&L Instruments) and data recording software (FluoMeasure, C&L Instruments). Thirty seconds of baseline fluorescence was recorded before stimulation of the vessels with either PE (1 or 10 μM) or ANG II (0.01 or 0.1 μM) for 1 min followed by agonist washout with MOPS buffer. The F₃₄₀-to-F₃₈₀ ratio (F₃₄₀/F₃₈₀) was calculated and used as relative indicator of cytosolic Ca²⁺ concentration. Because vessel diameter changes in response to agonist stimulation varied from vessel to vessel, the effects of PE and ANG II on Ca²⁺ flux were expressed as percent normalization to baseline. F₃₄₀/F₃₈₀ at baseline was expressed as 100%.

Quantitative real-time PCR analysis of ANG II type 1 receptors, AT₂Rs, and endothelial nitric oxide synthase expression levels in WT and Eln^+/− aortae and MAs.

Expression levels of ANG II type 1 (AT1) receptors (AT₁Rs), AT₂Rs, and endothelium nitric oxide (NO) synthase (eNOS) messages in aortae and MAs of WT and Eln^+/− mice were assessed by quantitative real-time PCR experiments. Tissue total RNA was isolated using the EZ Tissue RNA extraction kit (EZ BioResearch, St. Louis, MO) according to the manufacturer's instructions. The following primer pairs were used in the quantitative real-time PCRs as directed by the manufacturer (Life Technologies, New York, NY): AT₁R, primer 1 5′-TGCAGAATTATTCCTCTGCTCCT-3′ and primer 2 5′-TTCCTCAGGAGAAGGCTTGA-3′; AT₂R, primer 1 5′-AACTGGCACCAATGAGTCCG-3′ and primer 2 5′-CCAAAAGGAGTAAGTCAGCCAAG-3′; eNOS, primer 1 5′-CTGTGGTCTGGTGCTGGTC-3′ and primer 2: 5′-TGGGCAACTTGAAGAGTGTG-3′; and GAPDH, primer 1 5′-TGCACCACCAACTGCTTAG-3′ and primer 2 5′-GGATGCAGGGATGATGTTC-3′. The ΔΔC_t method (where C_t is threshold cycle) was used to calculate AT₁R, AT₂R, and eNOS mRNA expression after normalization to GAPDH expression.

Blood pressure and heart rate measurements.

Littermates of WT and Eln^+/− male mice of 2–4 mo of age were used for blood pressure and heart rate measurements. Baseline blood pressure was measured by placing a Millar pressure-transducing catheter in the right carotid artery under 1.5% isoflurane, as we have previously described (15). Fifteen minutes after jugular vein catheter implantation, baseline recordings were performed for 5 min followed by a bolus intravenous administration of the Rho kinase inhibitor Y-27632 (0.5, 1.0, and 1.5 mg/kg) with each dose in 10 μl of 0.9% saline. A 5-min period was allowed between injections and for blood pressure to reach steady state.

Data and statistical analyses.

Vasoconstriction was expressed as the percent change in vessel diameter from baseline according to the following formula: [(D_i − D)/D_i] × 100, where D_i is the initial diameter before application of the drug, and D is the measured diameter in the presence of the drug. Percent relaxation was expressed as the change in vessel diameter after preconstriction with PE according to the following formula: [(D − D_PE)/(D_i − D_PE)] × 100, where D_PE is the steady-state diameter after constriction with PE. Results are expressed as means ± SE. Data from multiple vessels of the same animal were averaged. pEC₅₀ was calculated by a computer-aided curve fit (SigmaPlot 11.0, Systat Software, San Jose, CA) using sigmoidal dose-response logistics. An unpaired Student's t-test and two-way ANOVA with repeated measures were used, where appropriate, to assess the effects of Eln hemizygosity on GPCR-induced vasoconstriction and endothelium-dependent and endothelium-independent vasodilatation of preconstricted MAs. Blood pressure variations before and after Y-27632 injections were compared using two-way ANOVA with repeated measures. A Newman-Keuls post hoc test was used to determine between-group differences. P values of <0.05 were considered statistically significant.

RESULTS

Effect of Eln hemizygosity on structural remodeling of aortae and MAs.

We have previously noted that Eln^+/− mice have thinner but more elastic lamellae and an increased number of smooth muscle layers in large elastic vessels compared with WT mice (15, 33). To determine how Eln insufficiency affects the resistance vasculature, we analyzed elastic lamellar units and smooth muscle layer thickness in the thoracic aorta and mesenteric resistance arteries by hematoxylin and eosin and Verhoeff-van Gieson staining. Consistent with our previous observations, we found that the elastic laminae were thinner and that there were ∼50% more elastic layers in the thoracic aorta of Eln^+/− relative to WT mice (data not shown). In second-order mesenteric resistance arteries, there were two layers of smooth muscle in both WT and Eln^+/− mice, but an extra discontinuous Eln layer was present between the smooth muscle cell layers in the middle of the Eln^+/− wall (Fig. 1). As in large vessels, the internal elastic lamina was thinner compared with WT animals. Wall thickness and luminal diameter, measured at 60 mmHg intraluminal pressure, were unchanged in Eln^+/− mice compared with WT mice (Table 1).

Fig. 1.

Representative photomicrographs of Verhoeff-van Gieson stains of small mesenteric arteries (MAs) of wild-type elastin (Eln^+/+; A) and Eln heterozygous (Eln^+/−; B) mice. An extra Eln-containing lamella in the Eln^+/− MA section is indicated by the arrowhead. Images of MA cross-sections (10 μm) were taken at ×40 magnification.

Table 1.

Structural properties of Eln^+/+ and Eln^+/− mesenteric arteries

Parameter	Eln+/+	Eln+/−
No. of arteries	14	12
Lumen diameter, μm	171 ± 15	193 ± 9
Outer diameter, μm	196 ± 16	216 ± 9
Wall thickness, μm	13.0 ± 0.8	11.2 ± 0.5
Cross-sectional area, ×1,000 μm2	7.9 ± 1.0	7.3 ± 0.6

Data are means ± SE. Eln^+/+, mice with wild-type elastin (Eln); Eln^+/−, Eln heterozygous mice.

Eln hemizygosity augments ANG II- and PE-induced vasoconstriction of small MAs.

To determine the physiological implications of Eln insufficiency, we measured GPCR-induced vasoconstriction and vasodilatation of small mesenteric resistance arteries from WT and Eln^+/− mice ex vivo. First, we determined the effects of Eln hemizygosity on vasoconstriction induced by two vasoconstrictors: PE and ANG II. PE-induced vasoconstriction is mediated by activation of α₁-adrenergic receptors coupled to G_q/11 class heterotrimeric G proteins, whereas ANG II-induced vasoconstriction is primarily mediated by activation of AT₁Rs coupled to G_q/11 and G_12/13 classes of G proteins (18, 22). As shown in Fig. 2A, PE-induced vasoconstriction was augmented at low to intermediate concentrations (1 and 10 μM) of the agonist. However, the vasoconstrictor response to the maximum concentration of PE was not different between WT and Eln^+/− MAs. In contrast to the effects of PE, ANG II-induced vasoconstriction was markedly augmented in Eln^+/− MAs compared with WT MAs (Fig. 2, B and C). Both the potency (pEC₅₀: 8.0 ± 0.2 in WT MAs vs. 8.6 ± 0.2 in Eln^+/− MAs, P < 0.05) and maximal vasoconstrictor response (efficacy) to ANG II (36 ± 4% contraction in WT MAs vs. 59 ± 5% constriction in Eln^+/− MAs, P < 0.01) were increased in Eln^+/− MAs compared with WT MAs. In contrast to the resistance vasculature, however, femoral arteries of WT and Eln^+/− mice responded similarly to ANG II (Fig. 2C), indicating that the augmented vasoconstriction is absent in conducting vessels.

Fig. 2.

Vasoconstrictor response of Eln^+/+ and Eln^+/− MAs to phenylephrine (PE) and ANG II. A: vasoconstrictor responses to PE expressed as mean percent decreases in diameter [n = 5–6 animals (2 vessels/animal) per group]. B: vasoconstrictor responses to ANG II expressed as mean percent decreases in diameter. C: vasoconstrictor responses of Eln^+/+ and Eln^+/− femoral arteries to ANG II. Data are expressed as means ± SE. *P < 0.05 and **P < 0.01, Eln^+/+ vs. Eln^+/−.

AT₂Rs contribute to the ANG II-induced contractile response in Eln^+/− MAs.

To elucidate the mechanisms mediating the augmented ANG II-elicited vasoconstriction of Eln^+/− MAs, we first used quantitative real-time PCR to measure AT₁R and AT₂R expression levels in the aorta and MAs. In the aorta, the expression of AT₁Rs and AT₂Rs was equivalent between WT and Eln^+/− mice (Fig. 3A); however, there was less AT₂R expression than AT₁R expression. In MAs, expression of AT₁Rs and AT₂Rs was decreased in Eln^+/− mice compared with WT mice (Fig. 3B). Taken together, these findings show that AT₁R and AT₂R expression are different in conducting versus resistance vessels at the mRNA level. Moreover, the data also indicate that the augmented ANG II-induced vasoconstriction is not due to an increased number of receptors for ANG II in Eln^+/− MAs compared with WT MAs.

Fig. 3.

Quantification of ANG II type 1 (AT₁R), ANG II type 2 receptor (AT₂R), and endothelial nitric oxide (NO) synthase (eNOS) mRNA levels in aortae (A) and MAs (B) of Eln^+/+ (n = 5) and Eln^+/− (n = 6) mice using quantitative real-time PCR. AT₁R, AT₂R, and eNOS mRNA expression levels were normalized to GAPDH mRNA levels in each sample. Data are expressed as means ± SE. *P < 0.05 vs. Eln^+/+ mice.

Several studies (19, 21, 36, 56) have established that AT₂R-mediated signaling opposes AT₁R-mediated effects in many physiological contexts, including AT₁R-mediated vasoconstriction and the AT₂R-mediated vasodilatory response in blood vessels. To test whether the augmented ANG II-induced vasoconstriction of Eln^+/− MAs is due to unopposed vasoconstriction resulting from decreased AT₂R-mediated vasodilation, we incubated vessels with the selective AT₂R antagonist PD-123319 (10 μM) before stimulation with ANG II. As shown in Table 2 and Fig. 4A, AT₂R blockade had no effect on the sensitivity (pEC₅₀) or maximal contractile response of WT MAs to ANG II. In contrast, AT₂R blockade reduced the sensitivity of Eln^+/− MAs to ANG II (Fig. 4B), suggesting that AT₂R stimulation contributes to the augmented ANG II-induced vasoconstriction of Eln^+/− resistance arteries. Stimulation with the AT₂R agonist novokinin caused a small but significant vasoconstrictor response in Eln^+/− MAs in a dose-dependent manner (Fig. 4C), further indicating that the augmented ANG II-evoked vasoconstriction is partly mediated by AT₂R activation.

Table 2.

Maximal contractile responses and pEC₅₀ of Eln^+/+ and Eln^+/− mesenteric arteries to ANG II in the absence or presence of the selective ANG type 2 receptor blocker PD-123319

	ANG II		ANG II + PD-123319
	Eln+/+	Eln+/−	Eln+/+	Eln+/−
No. of arteries	10	8	5	5
Maximal contractile response, %	36 ± 4	59 ± 5†	39 ± 5	53 ± 7
pEC50	8.1 ± 0.1	8.7 ± 0.1*	7.9 ± 0.2	8.2 ± 0.1‡

Data are means ± SE. pEC₅₀, sensitivity.

^*P < 0.05 and

^†P < 0.01 vs. Eln^+/+;

^‡P < 0.05 vs. Eln^+/− in the absence of PD-123319.

Fig. 4.

Vasoconstrictor responses of Eln^+/+ (A) and Eln^+/− (B) MAs to ANG II in the absence and presence of PD-123319 (10 μM). C: vasoconstrictor responses of Eln^+/+ and Eln^+/− MAs to the AT₂R agonist novokinin. Vessel responses are expressed as mean percent decreases in diameter [n = 4–6 animals (2 vessels/animal) per group]. Data are expressed as means ± SE. *P < 0.05 and **P < 0.01, vs. control or Eln^+/+.

PE-induced vasoconstriction of Eln^+/− resistance arteries is insensitive to Rho kinase inhibition.

ANG II induces vasoconstriction by activating downstream signaling pathways that are coupled to both G_q/11 and G_12/13 classes of G proteins. Because both PE- and ANG II-induced vasoconstriction were augmented in Eln^+/− MAs, we examined whether there was any alteration in Rho-Rho kinase signaling, a common effector system of G_q/11 and G_12/13 (see Fig. 9). To determine the effect of Eln hemizygosity on Rho-Rho kinase signaling-dependent vasoconstriction, PE- and ANG II-elicited contractile responses were performed in the presence of the specific Rho kinase inhibitor Y-27632. Inhibition of Rho kinase with increasing concentrations of Y-27632 decreased the maximal vasoconstrictor response to PE but not the sensitivity (pEC₅₀) of WT MAs (Fig. 5A and Table 3). In contrast, the contractile responses of Eln^+/− MAs to PE were insensitive to any concentration of Y-27632 (Fig. 5B and Table 3). Opposite to PE-induced vasoconstriction, ANG II-induced vasoconstriction of both WT and Eln^+/− MAs was reduced by Y-27632 (Fig. 5, C and D). Taken together, these results indicate that Eln insufficiency leads to insensitivity of Rho-Rho kinase signaling to the activation of G_q/11.

Fig. 9.

Model for potential signaling mechanisms mediating enhanced G protein-coupled receptor (GPCR)-induced vasoconstriction of resistance arteries from Eln^+/− mice. Vasoconstrictor agonists such as ANG II and PE activate GPCRs coupled to G_q/11 and/or G_12/13 classes of G proteins. Activation of G_12/13 can cause vasoconstriction by activating a GTP exchange factor (RhoGEF) that, in turn, activates RhoA-Rho kinase (RhoK), resulting in smooth muscle contraction. On the other hand, activation of G_q/11 triggers inositol 1,4,5-triphosphate (IP₃) release by phospholipase C (PLC)-β that, in turn, triggers the rise of intracellular Ca²⁺, leading to smooth muscle contraction. Vasoconstriction may also result from activation of the Rho-Rho kinase pathway after the activation of G_q/11 via an unknown protein(s). In Eln insufficiency, there is decreased endothelium-dependent regulation of G_q/11-evoked intracellular Ca²⁺ transients and uncoupling of G_q/11-RhoA-RhoK pathways, resulting in enhanced GPCR-induced vasoconstriction.

Fig. 5.

Effect of Rho kinase inhibition on contractile responses of Eln^+/+ (n = 6) and Eln^+/− (n = 7) MAs to PE (A and B) and ANG II (C and D). Resistance arteries were incubated in the absence or presence of the Rho kinase inhibitor Y-27632 at various concentrations (0.1, 1.0, or 5 μM) for 20 min before stimulation with increasing concentrations of ANG II or PE. Data are expressed as means ± SE. **P < 0.01 vs. control. NS, not significant.

Table 3.

pEC₅₀ values of Eln^+/+ and Eln^+/− mesenteric arteries to ANG II and PE in the presence or absence of the Rho kinase inhibitor Y-27632

	ANG II		PE
	Eln+/+	Eln+/−	Eln+/+	Eln+/−
No. of arteries	4–10	5–8	5–7	5–8
Y-27632
0 μmol/l	8.0 ± 0.2	8.6 ± 0.1*	5.7 ± 0.1	5.6 ± 0.2
0.1 μmol/l	8.1 ± 0.1	8.1 ± 0.2	5.6 ± 0.6	6.2 ± 0.2
1 μmol/l	8.5 ± 0.5	7.9 ± 0.2†	5.6 ± 0.1	6.1 ± 0.2
5 μmol/l	ND	7.4 ± 0.3†	5.0 ± 0.6	5.7 ± 0.1

Data are means ± SE. PE, phenylephrine; ND, not determined.

^*P < 0.05 vs. Eln^+/+;

^†P < 0.01 vs. Eln^+/− in the absence of Y-27632.

Rho kinase inhibition normalizes blood pressure in Eln^+/− mice.

In our previous study (15), we showed that hypertension in Eln^+/− mice involves enhanced activity of the renin-angiotensin system (RAS), since nonselective blockade of ANG II receptors with saralasin lowered the blood pressure of Eln^+/− but not WT control mice. In the present study, we determined whether augmented Rho-Rho kinase signaling contributes to the elevated blood pressure in Eln^+/− mice. As previously reported (15), baseline systolic blood pressure was elevated in Eln^+/− mice compared with WT control mice (106 ± 2 mmHg in WT mice vs. 123 ± 5 mmHg in Eln^+/− mice, P < 0.01; Fig. 6A). Intravenous administration of 0.5 mg/kg of the Rho kinase inhibitor Y-27632 decreased systolic blood pressure in Eln^+/− mice to a level comparable to the baseline of WT control mice. The subsequent administration of higher doses of Y-27632 (1.0 and 1.5 mg/kg) caused further reductions in blood pressure in both genotypes. Baseline heart rate was similar at baseline and increased slightly at all doses in both groups (Fig. 6B). Taken together, these results indicate that the elevated blood pressure in Eln^+/− mice is mediated in part by augmented ANG II-mediated activation of Rho-Rho kinase signaling.

Fig. 6.

Effect of the Rho kinase inhibitor Y-27632 (0.5, 1.0, and 1.5 mg/kg) on systolic blood pressure (SBP; A) and heart rate [HR; in beats/min (bpm); B] of Eln^+/+ (n = 8) and Eln^+/− (n = 7) mice. Data are expressed as means ± SE. *P < 0.05 and **P < 0.01 vs. the corresponding baseline; ##P < 0.01 vs. Eln^+/+ mice at baseline.

GPCR-evoked cytosolic Ca²⁺ flux is augmented in Eln^+/− resistance arteries.

To determine whether a change in G_q/11-dependent Ca²⁺ mobilization is involved in the augmented vasoconstriction of Eln^+/− MAs, we measured PE- and ANG II-induced intracellular Ca²⁺ fluxes using the fura-2 fluorescence ratio (F₃₄₀/F₃₈₀). In WT MAs, the application of 1 μM PE resulted in a small change (∼3% above baseline) in F₃₄₀/F₃₈₀, whereas the same concentration of PE caused a marked increase in F₃₄₀/F₃₈₀ in Eln^+/− arteries (Fig. 7A). The application of a higher concentration of PE (10 μM) in WT vessels caused a further increase in F₃₄₀/F₃₈₀, indicating that the low F₃₄₀/F₃₈₀ response to 1 μM PE was not due to poor fura-2 dye loading (Fig. 7B). As shown in Fig. 7, C and D, the application of ANG II (0.01 and 0.1 μM, respectively) also elicited an intracellular Ca²⁺ flux that was markedly higher in Eln^+/− MAs compared with WT MAs. Taken together, these results indicate that activation of GPCRs coupled to G_q/11 results in augmented intracellular Ca²⁺ mobilization in Eln-insufficient resistance arteries.

Fig. 7.

Percent fura-2 ratio [fluorescence ratio at 340 to 380 nm (F₃₄₀/F₃₈₀)] of Eln^+/+ (n = 6) and Eln^+/− (n = 8) MAs induced by the application of PE (1 and 10 μM; A and B) and ANG II (0.01 and 0.1 μM; C and D). F₃₄₀/F₃₈₀ at baseline is expressed as 100%. Data are expressed as means ± SE.

Endothelium-dependent vasodilation is impaired in Eln^+/− resistance arteries.

We (15) have previously reported that ACh-induced vasodilation is impaired in the Eln^+/− renal artery but not in the descending aorta or carotid artery, suggesting that the vasodilation defect in Eln^+/− vessels is more prominent in small arteries that have less Eln. Accordingly, we assessed vasodilatation induced by endothelium-dependent and endothelium-independent mechanisms in preconstricted second-order MAs from WT and Eln^+/− mice. We found that Eln^+/− MAs showed remarkable impairment of ACh-evoked vasodilatation, exhibiting only ∼25% of the vasodilatory response seen for WT MAs at the maximum dose of ACh. However, there was no difference in eNOS message levels in MAs from WT and Eln^+/− mice (Fig. 3B). Moreover, endothelium-dependent vasodilation in Eln^+/− MAs was sensitive to inhibition of eNOS with N-nitro-l-arginine methyl ester (l-NAME) and to inhibition of EDHF with apamin + TRAM-34 (Fig. 8, A and B).

Fig. 8.

Vasodilatory responses of Eln^+/+ and Eln^+/− MAs. A and B: concentration response of ACh-induced vasodilation in the absence or presence of N-nitro-l-arginine methyl ester [l-NAME; 100 μM, n = 5–6 animals (2 vessels/animal) per group; A] or the EDHF inhibitors apamin (100 nM) + TRAM-34 (1 μM) (B). C and D: concentration responses of sodium nitroprusside (SNP)- and isoproterenol (ISO)-induced vasodilatation. E: concentration response of ACh-induced vasodilation of Eln^+/+ and Eln^+/− MAs in the absence or presence of Mn(III)tetrakis(4-benzoic acid)porphyrin chloride [MnTBAP; 100 μM, n = 3–5 animals (2 vessels/animal) per group]. F: concentration response of ACh-induced vasodilation of Eln^+/+ and Eln^+/− femoral arteries. Data shown are percent changes in vessel diameter ± SE. **P < 0.01, Eln^+/+ vs. Eln^+/−; #P < 0.05 and ++,##P < 0.01 vs. corresponding L-NAME- or EDHF-treated; $P < 0.05 and $$P < 0.01, Eln^+/+ MnTBAP vs. Eln^+/− MnTBAP.

To determine whether the impaired endothelium-dependent vasodilation was due to a defect in the smooth muscle intrinsic relaxation mechanism, we evaluated endothelium-independent vasodilatation elicited by the exogenous NO donor SNP and by the nonselective β-adrenergic receptor agonist ISO. As shown in Fig. 8, C and D, WT and Eln^+/− MAs showed a similar vasodilatory response to increasing concentrations of SNP and ISO. To further assess whether the impaired endothelial function might be due to decreased bioavailability of EDRFs as a result of increased ROS levels, endothelium-dependent vasodilation was evaluated in the presence of the cell-permeable superoxide dismutase mimetic MnTBAP (100 μM). As shown in Fig. 8E, vessel treatment with the ROS scavenger did not have any effect on the impaired endothelium-dependent vasodilation of Eln^+/− MAs. In contrast, femoral arteries from WT and Eln^+/− mice showed a similar vasodilatory response to ACh (Fig. 8F). Taken together, these results indicate that the impaired ACh-induced vasodilatation of Eln^+/− MAs is due to a defect in the vascular endothelial function of the resistance vasculature.

DISCUSSION

Eln insufficiency results in thinner elastic lamellae and the formation of extra Eln and smooth muscle layers (lamellar units) in large conduit arteries in humans and mice (51, 52). In mice, these structural changes are accompanied by a 15- to 30-mmHg increase in arterial blood pressure compared with WT control mice (15, 28, 50). While these structural changes clearly influence cardiovascular function, it is doubtful that they alone explain the hypertension associated with low Eln levels. Here, we present data showing that functional changes in the resistance vasculature are consistent with augmented peripheral resistance and elevated blood pressure in mice with Eln haploinsufficiency. Assessment of vascular reactivity to GPCR agonists revealed that Eln^+/− MAs have an augmented vasoconstrictor response to ANG II and PE and a marked impairment in ACh-evoked, endothelium-dependent vasodilatation. These functional changes in the resistance vasculature resulting from Eln insufficiency contribute to the development of hypertension since blood pressure can be normalized by an inhibitor that targets signaling downstream of vasoactive receptors.

Total peripheral vascular resistance is regulated by the balance between constriction and dilation mainly of resistance arteries in the microcirculation. Augmented peripheral resistance may result from 1) an impairment of the mechanisms mediating vasodilation, thereby leading to unopposed vasoconstriction; 2) aberrant vasoconstrictor mechanisms that lead to hypercontractility that overcomes vasodilatory tone; or 3) an impairment in both constrictor and dilatory mechanisms that result in hypercontractility. Vasoconstriction induced by ANG II is mediated by signaling pathways downstream of G_q/11 and G_12/13 (22, 35). Both classes of G proteins can be activated by the AT₁R. AT₁R-dependent activation of G_q/11 induces smooth muscle contraction by activating a downstream signaling cascade involving phospholipase-β1-mediated production of inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol, stimulation of Ca²⁺ release from intracellular stores by IP₃, and activation of the actomyosin contractile apparatus by Ca²⁺/calmodulin-activated myosin light chain kinase (23, 24, 46, 48). On the other hand, AT₁R-dependent activation of G_12/13 induces smooth muscle contraction by the Rho-Rho kinase signaling pathway that inhibits myosin phosphatase, leading to prolonged myosin phosphorylation and Ca²⁺ sensitization of the actomyosin contractile apparatus (18, 35, 46, 57). Vasoconstrictor signaling by G_q/11 and G_12/13 are not mutually exclusive, as work by Somlyo and others (17, 37, 58) have shown that Rho-Rho kinase signaling can be triggered by activation of G_q/11 in the vascular smooth muscle with p63 Rho guanine nucleotide exchange factor (p63RhoGEF) serving as the molecular switch. GPCR agonist-dependent hypercontractility of resistance arteries is a hallmark of hypertension in humans and animal models. In addition to resistance artery hyperacontractility accompanied by endothelial dysfunction characterized by impaired endothelium-dependent vasodilation, defects in regulatory mechanisms and/or increased activity of signaling pathways intrinsic to vascular smooth muscle play a key role in the augmentation of peripheral resistance.

Several lines of evidence in this study indicate that a combination of increased vasoconstriction and impaired endothelium-dependent vasodilation of the resistance vasculature is involved in pathogenesis of hypertension in Eln insufficiency. First, vasoconstriction induced by PE and ANG II is augmented. Second, endothelium-dependent vasodilation is markedly impaired. Third, both vasoconstriction and vasodilation are normal in conductance arteries. Fourth, intravenous administration of a Rho kinase inhibitor reduces blood pressure of Eln^+/− mice to the level of WT control mice. In agreement with our previous study, the present findings also show that the RAS plays a primary role in the mechanisms underlying the augmented vasoconstriction in Eln^+/− mice. In the intact mouse, nonselective blockade of ANG II receptors with saralasin or selective blockade of AT₁Rs with candesartan normalized blood pressure in Eln^+/− mice, indicating that increased ANG II effects contribute to the hypertension in Eln^+/− mice (15). However, it was not clear whether such an effect of the RAS on blood pressure was due merely to increased circulating levels of ANG II or abnormalities in the signal transduction pathways mediating the effect of ANG II on blood pressure. We have addressed this question in this study by first assessing the reactivity of both resistance and conduit arteries to vasoconstrictor agonists (PE and ANG II) that stimulate G_q/11 and G_12/13 classes of G proteins. The vasoconstrictor response of Eln^+/− arteries is augmented; however, this effect becomes more apparent with decreasing vessel diameter, as evidenced by reactivity of the aorta and carotid and renal arteries in our previous study (15) and femoral arteries and small MAs in this study. Furthermore, changes in the structural and mechanical properties of blood vessels of Eln-insufficient mice and patients with WBS are more prominent in conduit vessels. These structural changes extend into the resistance arteries in adult mice, as we have shown in this study. However, the accompanying functional alteration is limited to the resistance arteries, which raises the possibility that these changes are triggered during postnatal maturation of the circulatory system when Eln levels plateau and vascular stiffness becomes more pronounced. In accordance with this hypothesis, a previous study by Le et al. (30) showed that decreased diameter and compliance as well as increased stiffness of the great arteries occur before the elevation in systolic blood pressure, which begins to rise by day 14 after birth in Eln^+/− mice. Another mechanism that could mediate the augmented vasoconstriction and blood pressure in Eln^+/− mice is increased transduction of the ANG II signal due to increased receptor expression, thereby amplifying the magnitude and frequency of G protein activation. However, this possibility can be ruled out by the finding that mRNA expression of both ANG II receptor subtypes in Eln^+/− MAs is decreased compared with levels in WT MAs.

The decreased AT₂R level in Eln^+/− MAs suggests that the augmented ANG II-induced constriction could result from unopposed AT₁R-mediated vasoconstriction. Several studies (54–56) have indicated that the AT₂R activates eNOS via the kinin-kallikrein pathway, leading to NO generation in the endothelium that subsequently activates smooth muscle intrinsic relaxation mechanisms via the cGMP-PKG pathway. Thus, it would be expected that reduced expression or blockade of AT₂Rs would lead to augmented ANG II-induced vasoconstriction. Interestingly, we found that an AT₂R antagonist decreased ANG II-induced vasoconstriction of Eln^+/− MAs, suggesting that the augmented vasoconstriction of Eln^+/− resistance arteries is in part due to the activation of AT₂Rs. The contractile phenotype mediated by AT₂Rs was unexpected since AT₂R activation has been shown to counteract the vasoconstrictor effect of AT₁Rs by inducing vasodilatation (19, 21, 36, 56). However, in WT MAs, blockade of AT₂Rs had no effect on ANG II-induced vasoconstriction, suggesting that AT₂R-mediated vasodilatation is limited or nonexistent in this vascular bed. Thus, the contractile effect of AT₂Rs in Eln^+/− MAs cannot be attributed to a defect in AT₂R-mediated vasodilatation. Furthermore, because the AT₂R message level was reduced in Eln^+/− MAs, it is less likely that the ANG II-induced contractile response mediated by AT₂Rs is due to increased receptor expression. PD-123319 has a low binding affinity for AT₁Rs at concentrations of 10 μM and above (5). However, the possibility that the decreased ANG II-induced vasoconstriction by PD-123319 was due to effects of the antagonist at AT₁Rs was ruled out because activation of AT₂Rs by the selective agonist novokinin evoked a contractile response in Eln^+/− MAs but not in WT MAs. This novel finding reveals a previously unknown mechanism by which the RAS is involved in resistance vascular function defects associated with hypertension in Eln insufficiency.

Our findings suggest a potential mechanism whereby the AT₂R elicits a contractile response. AT₂Rs could elicit vasoconstriction by heterodimerizing with AT₁Rs, as previously demonstrated in PC12 cells, fibroblasts, and myometrial tissues (1, 2). This hypothesis is consistent with observations showing that AT₂R blockade decreased the pEC₅₀ of ANG II-induced constriction in Eln^+/− MAs to the level of WT control MAs. Alternatively, AT₂Rs could induce vasoconstriction via an unidentified signaling pathway that is independent of the Rho-Rho kinase signaling pathway, since a noticeable ANG II-induced vasoconstriction persisted in Eln^+/− MAs after Rho kinase blockade by a concentration of the inhibitor that almost completely blocks vasoconstriction in WT control MAs. Validating the contractile and pressor effect of AT₂R antagonists in live animals would be difficult because the vasodilatory response of the AT₂R action in large vessels will likely mask changes in the microcirculation.

ANG II-induced vasoconstriction in MAs and baseline blood pressures of WT and Eln^+/− mice were sensitive to Rho kinase inhibition, indicating that the reliance of G_12/13-coupled, AT₁R-evoked contraction on Rho signaling and its role in blood pressure regulation are not suppressed by Eln insufficiency. However, the results also indicate that this signaling pathway is involved in the development of hypertension in Eln^+/− mice, since baseline systolic blood pressure was reduced to WT control mice after acute injection of the Rho kinase inhibitor Y-27632. In contrast, there is a dramatic difference in the effect of Rho kinase inhibition on α₁-adrenergic receptor-induced vasoconstriction between MAs of WT and Eln^+/− mice. In wild-type WTs, Rho kinase inhibition decreases the maximal vasoconstriction triggered by G_q/11-coupled α₁-adrenergic receptors. As would be expected of an effect downstream of the receptor, Rho kinase inhibition has no effect on the pEC₅₀ of PE-induced vasoconstriction. Thus, in wild-type small mesenteric resistance arteries, adrenergic receptor-evoked vasoconstriction is mediated in part by the G_q/11-Rho-Rho kinase signaling axis. This signaling axis is either masked or abolished as a result of Eln insufficiency, since Rho kinase inhibition in MAs from Eln^+/− mice had no effect on PE-induced vasoconstriction.

The G_q/11-mediated rise in intracellular Ca²⁺ concentration ([Ca²⁺]_i) triggered by PE or ANG II was much greater in Eln^+/− MAs compared with WT control MAs. One interpretation of this outcome is that a component of the functional remodeling in Eln^+/− resistance arteries is increased Ca²⁺ sensitization. In the case of ANG II receptor-induced vasoconstriction of Eln^+/− MAs, this interpretation is likely, since there is a positive correlation between augmented [Ca²⁺]_i and ANG II-induced vasoconstriction. In contrast, there is a dissociation between augmented [Ca²⁺]_i and vasoconstriction induced by α₁-adrenergic receptor stimulation. Based on the level of the PE-induced rise in [Ca²⁺]_i, a much stronger vasoconstriction would be expected than what was observed in this study. Such a disparity between GPCR-induced [Ca²⁺]_i and smooth muscle contraction can occur in arterial smooth muscle cells (2, 16, 26, 27, 47). The proposed mechanisms to account for the [Ca²⁺]_i-contraction dissociation is the existence of a noncontractile Ca²⁺ compartment, where Ca²⁺ levels may rise due to activation of different Ca²⁺ entry pathways without causing increased myosin light chain phosphorylation and subsequent smooth muscle contraction (3, 4, 25). The approach that was used to assess GPCR-induced Ca²⁺ influx (the Ca²⁺ indicator fura-2) detects overall cytosolic Ca²⁺ and is therefore inadequate to resolve potential differences in the Ca²⁺ concentration in contractile and noncontractile compartments and changes that may occur in smooth muscle cells from Eln-insufficient mice. Future exploration of this mechanism may require the use of compartment-specific Ca²⁺ indicators.

Our present findings also indicate that defects in GPCR-dependent vasodilation contribute to increased vasoconstriction of resistance arteries from Eln^+/− mice. We (15) have previously observed that ACh-induced vasodilation is reduced in small conduit arteries, leading us to speculate that Eln insufficiency causes an endothelium-dependent vasodilation defect that is prominent in the microcirculation. This possibility was confirmed by the finding that ACh-induced vasodilation was markedly impaired in small MAs from Eln^+/− mice. In resistance arterioles, endothelium-dependent vasodilation is mediated by the endothelium-derived relaxing factors NO and EDHF (9, 41). The dependence of GPCR-induced vasodilation on these relaxing factors can be ascertained using well-characterized pharmacological inhibitors (l-NAME and apamin + TRAM-34) that can selectively block NO- and EDHF-dependent vasodilation, respectively (7, 14, 53). Thus, we used these tools to further unravel the mechanisms mediating the vasodilation defect of mesenteric resistance arteries from Eln^+/− mice. Our findings indicate that despite attenuated vasodilation, the presence of either l-NAME or apamin + TRAM-34 further reduced ACh-induced vasodilation of MAs from Eln^+/− mice, indicating that the endothelium-dependent vasodilation is attenuated but not completely abolished in Eln insufficiency. We ruled out the possibility that impaired ACh-induced vasodilation is due to defects in smooth muscle-intrinsic relaxation mechanisms activated by the NO-cGMP or adenylyl cyclase-cAMP pathway (18, 43) because MAs from Eln^+/− mice showed the same level of vasodilation as WT control MAs when challenged with an exogenous NO donor or β-adrenergic receptor agonist. How Eln insufficiency causes decreased levels of EDRFs leading to endothelial dysfunction remains to be determined. However, one potential mechanism that likely mediates endothelial dysfunction in Eln^+/− resistance arteries is increased ROS levels. It is well documented that ROS generation from NADPH oxidases is an important factor in ANG II-induced hypertension (13, 40, 44). Furthermore, in both small and large vessels, ANG II is known to increase the expression of Nox1 and Nox2, which are components of NADPH oxidase. Increased ROS produced by Nox1 and Nox2 enzymes is responsible for endothelial dysfunction via the superoxide-mediated inactivation of the endothelium-derived vasodilator NO (29). Thus, because Eln^+/− mice have elevated ROS in conduit vessels (28), the impaired ACh-induced vasodilation in Eln^+/− MAs might be partly due to ROS inactivation of NO. However, our findings indicate that increased ROS activity does not to play a role in the impaired endothelium-dependent vasodilation or augmented vasoconstriction of Eln^+/− MAs, since treatment with the ROS scavenger did not improve ACh-induced vasodilation or ANG II-evoked vasoconstriction (data not shown). Because superoxide dismutase scavenges only superoxide, it still remains to be determined whether other ROS members (such as hydrogen peroxide and peroxynitrite) may be involved in the impaired relaxation. It is also possible that activated or receptor-mediated levels rather than baseline ROS levels are augmented in Eln^+/− arteries, which may not have been detected using our present experimental paradigm wherein basal ROS levels were acutely targeted.

The structural changes that occur in MAs in response to Eln haploinsufficiency are different than those occurring in the large conducting vessels. Common to both large and small Eln^+/− vessels are thinner elastic lamellae, but the lamellar number in large vessels increases as a consequence of additional smooth muscle cell layers that form in the vessel wall. In Eln^+/− MAs, the number of smooth muscle cell layers is equivalent to WT MAs, but a discontinuous band of Eln appears between the existing two smooth muscle cell layers. Another difference relates to wall thickness, which is decreased in Eln^+/− conducting vessels at mean physiological pressure compared with WT conducting vessels but does not change in Eln^+/− MAs. The changes in Eln^+/− wall structure are also different from those that occur in the resistance vasculature of rodents exposed to chronic hypertension outside of Eln insufficiency (20, 38, 39). Although these alterations are not well understood, previous reports (2, 38, 39) have shown that they involve increased (hypertrophy), decreased (hypotrophy), or rearranged (eutrophy) arterial wall components, particularly the smooth muscle cell layers and extracellular matrix proteins. Indeed, MAs from spontaneously hypertensive rats undergo eutrophic remodeling involving the reorganization and thickening of Eln in the internal elastic lamina (6). This is opposite to what is seen in Eln^+/− MAs, where both the internal and external elastic layers are thinner than those in WT MAs.

In conclusion, our results support a model of altered endothelial and smooth muscle function in the resistance vasculature that results in augmented vascular tone, which potentially involves downregulation or loss of an unknown regulatory protein(s) in smooth muscle leading to uncoupling of G_q/11 and Rho-Rho kinase signaling pathways and an augmented GPCR-evoked rise in intracellular Ca²⁺ that, together, contribute to the high blood pressure in Eln^+/− mice (Fig. 9). The functional changes in vessel reactivity reflect a permanent change in the homeostatic state of the resistance vasculature in response to chronic exposure to high ANG II levels associated with Eln insufficiency. We suggest that these functional alterations and those previously reported for large arteries are part of an important adaptive mechanism for maintaining tissue perfusion and cardiovascular function when elastic levels are lower than normal (15). This model is supported by the observed changes in GPCR-induced dilation and constriction, both of which favor augmented vascular tone and increased blood pressure. Thus, modulating GPCR-dependent regulation of vascular resistance may provide a novel means of controlling high blood pressure in diseases that are associated with Eln insufficiency or changes in the vascular extracellular matrix.

GRANTS

This work was supported by National Institutes of Health Grants HL-075632 and GM-445920 (to K. J. Blumer), NS-30555, HL-57540, and NS-32636 (to H. H. Dietrich), HL-53325, HL-74138, and HL-105314 (to R. P. Mecham) and American Heart Association Postdoctoral Fellowship 09POST2260099 (to P. Osei-Owusu).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: P.O.-O., K.B., and R.P.M. conception and design of research; P.O.-O., R.H.K., and K.B. performed experiments; P.O.-O., R.H.K., and R.P.M. analyzed data; P.O.-O., H.H.D., K.B., and R.P.M. interpreted results of experiments; P.O.-O. and R.P.M. prepared figures; P.O.-O., K.B., and R.P.M. drafted manuscript; P.O.-O., B.A.K., H.H.D., K.B., and R.P.M. edited and revised manuscript; P.O.-O. and R.P.M. approved final version of manuscript.