Elastin (ELN), the main component of elastic fibers, is responsible for conduit arteries' elastic recoil. This recoil is necessary to dampen the pulsatile flow of ventricular ejection at the level of the ascending aorta and transform it into continuous flow at the level of arterioles or small resistance arteries. Elastic fibers are organized into fenestrated concentric sheets or lamellae in blood vessels. Decreased elasticity of large arteries with aging is attributed to fragmentation and thinning of these lamellae and results in increased pulse wave velocity leading to a greater augmentation of the central aortic systolic and pulse pressures (1, 2). Similarly, genetic reduction of elastin through deletion of a single copy of the gene ELN (supravalvular aortic stenosis—SVAS, OMIM #185500) or deletion of ELN as part of a 25–27 coding gene microdeletion of chromosome 7 (Williams syndrome, OMIM #194050) leads to increased pulse wave velocity and hypertension (3–5).

Similar to humans with SVAS and Williams syndrome, mice hemizygous for the elastin gene (Eln^+/−) develop large artery stiffness and systolic hypertension (6, 7). Interestingly, the increased large artery stiffness in Eln^+/− mice precedes the appearance of hypertension (8) and is not affected by commonly used anti-hypertensives (9). Increased large artery stiffness and central systolic and pulse pressures often lead to structural and functional changes in small resistance arteries that further exacerbate hypertension and a vicious cycle ensues (10). This appears to be the case in elastin insufficiency as recent studies showed altered resistance vessel reactivity that is vascular bed-specific (11–13). Mesenteric arteries (MAs) and middle cerebral arteries (MCAs), but not gastrocnemius feed arteries (GFAs), were found to have impaired endothelial-dependent dilation to acetylcholine due to decreased nitric oxide availability resulting from increased oxidative stress (11, 12, 14). Furthermore, MAs and MCAs, but not GFAs, had an increased contractile response to angiotensin II (ang II) (11, 13). Interestingly, the hypercontractile response of mesenteric arteries to ang II was mediated, at least in part, by the angiotensin II type 2 receptor (AT₂R) as blockade of AT₂R with the antagonist PD123319 decreased the contractile response of MAs to ang II while its activation with novokinin resulted in vasoconstriction (11).

Given the multitude of evidence suggesting a vasodilatory role for AT₂R particularly in disease states and the consideration for the use of AT₂R agonists for patients with COVID-19 and idiopathic pulmonary fibrosis among others (clinicaltrials.gov), we sought to determine the cardiovascular role of AT₂R in elastin insufficiency in vivo. We bred elastin insufficient (Eln^+/−) mice to AT₂R knock-out (Agtr2^−/Y) mice and examined cardiovascular endpoints. While loss of AT₂R did not affect large artery structure or function, it lowered blood pressure in elastin insufficient mice, suggesting that AT₂R plays a vasocontractile role in elastin insufficiency. This observation has significant therapeutic implications since AT₂R agonists, which may be beneficial in some conditions such as stroke (15, 16), aneurysm formation (17, 18) and myocardial fibrosis (19), would not be appropriate in patients with elastin insufficiency.

Ang II, the principal effector of the renin-angiotensin system, exerts its functions in physiological and pathological states mainly through two receptors, AT₁R and AT₂R. In hypertension, the pathologic remodeling that occurs, including vasoconstriction, fibrosis, proliferation, and inflammation, has been attributed to ang II's actions through AT₁R. Over the past two to three decades, a great deal of effort has focused on understanding the role of the more elusive AT₂R. Evidence suggests that while its levels are low in the adult cardiovascular system at baseline, AT₂R expression increases significantly in pathological conditions and it is thought to counter-balance the effects of ang II by promoting a vasodilatory, anti-fibrotic, apoptotic, and anti-inflammatory phenotype (23, 24). Often the vasodilatory effect of AT₂R is only evident when the vasoconstrictor action of AT₁R is blocked. At baseline, AT₂R knock-out mice were normotensive but showed an increased pressor response to ang II infusion (20). With the availability of several non-peptide AT₂R agonists, their use is being investigated as a potential therapeutic option in several disease conditions. In this report, based on in vitro data suggesting a vasocontractile role for AT₂R in elastin insufficiency, we sought to determine whether AT₂R contributes to elastin insufficiency-mediated hypertension in vivo. Using mouse models with genetic loss or insufficiency of AT₂R and ELN, we show that, unlike its protective role in heart failure, myocardial infarction and aneurysms, in the context of elastin insufficiency loss of AT₂R improves blood pressure making its activation a potentially detrimental therapeutic strategy in this disease state.

While initially surprising, the observation that AT₂R may play a vasocontractile role has been made in other models of hypertension. For instance, Touyz et al. (25) showed an enhanced contractile response to ang II in mesenteric arteries from spontaneously hypertensive rats (SHR) compared to Wystar-Kyoto rats (WKY). This response was reduced by AT₂R blockade in young but not old SHR. Similarly, coronary arteries from SHR were found to have enhanced constriction to ang II, that was attributed to the absence of counter-regulatory AT₂R-mediated relaxation and/or a change in the AT₂R phenotype from dilatory to contractile (26).

An interesting observation from our study is that loss of AT₂R decreases both systolic and diastolic blood pressure, while pulse pressure, an indicator of conduit artery stiffness, remains significantly elevated in Agtr2^−/y;Eln^+/− mice compared to WT and Agtr2^−/y mice. These data support the large artery pressure-diameter measurements showing that loss of AT₂R does not affect large artery mechanics in elastin insufficiency. Rather, loss of AT₂R likely affects peripheral vascular resistance leading to a reduction in both systolic and diastolic blood pressure.

Activation of AT₂R by ang II has been shown to increase nitric oxide (NO) production, which activates guanylate cyclase to generate cyclic guanosine monophosphate (cGMP) leading to vasodilation (27, 28). The mechanism by which AT₂R leads to vasoconstriction in elastin insufficiency is unclear at this time. Similar to WT, Agtr1 is expressed at higher levels than Agtr2 in Eln^+/− vessels, and both Agtr1 and Agtr2 expression levels were unchanged in aortae and reduced in mesenteric arteries of Eln^+/− mice (11), making relative changes in receptor levels an unlikely explanation for the observed blood pressure response. It is interesting to note however that, while usually thought of as monomers, G protein-coupled receptors like AT₁R, AT₂R and bradykinin receptor (B₂R) have been shown to heterodimerize and adopt either an enhanced or an altered function. For instance, heterodimerization of AT₁R and B₂R led to increased activation of Gαq and Gαi, the two major signaling proteins activated by AT₁R (29). This AT₁R-B₂R heterodimerization was shown to contribute to ang II hypersensitivity in pre-eclampsia (30). AT₂R has been shown to dimerize with B₂R leading to enhanced NO and cGMP (27). Since AT₂R expression was reduced in elastin insufficient mesenteric arteries (11), it is interesting to speculate that AT₂R-B₂R dimer formation may be affected, or alternatively, that AT₂R heterodimerizes with AT₁R in elastin insufficiency, resulting in vasoconstriction rather than vasodilation; hypotheses that will be the focus of future investigation.

In summary, using a mouse model of elastin insufficiency-mediated hypertension, here we show that loss of AT₂R improves blood pressure in this model. While the process of elastin insufficiency is distinct, with normal aging older adults develop vascular elastic fiber thinning, systolic hypertension with widened pulse pressure and large artery stiffness, all characteristics of elastin insufficient mice. Therefore, if AT₂R agonists are to be considered for clinical use, carefully designed randomized clinical trials with special attention to patient population and endpoints will be necessary to ensure that they are not contributing to disease, particularly hypertension. AT₂R agonists will likely be useful in a context-specific manner.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by Institutional Animal Care and Use Committee of Washington University School of Medicine.

RM, BK, and CH conceived the study design. ML, RR, and CH performed experiments, generated and analyzed data. CH drafted the manuscript. All authors have read and approved the final manuscript.

This work was supported by National Institutes of Health grants K08 HL135400 to CH and R01-HL53325 to RM. Funding for BK came from the Division of Intramural Research of the NIH, ZIA HL006210. Funds were also provided by the Ines Mandl Research Foundation to RM.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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