BEST3-mediated MEKK2/3 Activation - A Novel Therapeutic Target in Aortopathies

Aortopathies are life-threatening vascular diseases that have a spectrum of manifestations, including aneurysm, dissection, and rupture. Currently, surgical approaches are the only treatment for this disease. Therefore, there is an urgent need to develop new therapeutic options to treat and prevent aortopathies and their devastating consequences. Smooth muscle cells (SMC), the primary cell type in the media layer, exert a crucial role in the structural stability of the aortic wall. In the current issue of Circulation, Zhang, et al¹ delved into the molecular basis of aortopathies, with a particular focus on bestrophin 3 (BEST3) regulation of mitogen-activated kinases (MAPKs) in SMCs to propose a new therapeutic approach to attenuate these diseases.¹

Bestrophin is a superfamily of four isoforms of Ca²⁺-activated Cl⁻ channels, predominantly expressed in the brain, eyes, and gastrointestinal tract.² BEST3 is also expressed in aortic SMCs. Recent studies have revealed its contribution to SMC apoptosis and intimal inflammation in the aorta, in addition to its role as an anion channel.^3,4 Zhang, et al explored the role of BEST3 in SMCs in aortopathy formation using multiple approaches that encompassed human tissues and mouse models of aortopathies.¹ First, the authors deleted BEST3 in SMCs using Tagln-Cre (SMC-BEST3−/−) and observed decreased survival due to aortic rupture in aged mice. SMC-BEST3−/− mice that survived the 72 weeks of observation had severe dilatation in the ascending, arch, descending thoracic, and supra-renal abdominal aortic regions. SMC-BEST3 deficiency in 8-week-old mice also augmented aortic rupture and aneurysms during 4 weeks of angiotensin II (AngII)-infusion. Conversely, overexpression of BEST3 using adenovirus or lentivirus ameliorated AngII-induced aortic rupture and aneurysm formation. BEST3 deletion in endothelial cells did not show discernable aortic pathologies. These data support the notion that BEST3 in SMCs exerts a protective role in maintaining the integrity of the aortic wall. To determine the molecular basis of aortic pathologies by SMC-BEST3−/−, single-cell RNA sequencing (scRNAseq) was performed. Aortic SMCs had four sub-clusters that were defined as contractile, proliferating, stressed, and fibroblast-like SMCs. SMC-BEST3−/− decreased the number of fibroblast-like SMCs that had the most abundant mRNA for extracellular matrix (ECM) components, such as Lum and Dcn. Co-immunoprecipitation, coupled with mass spectrometry, identified the MEKK3-MAPKs pathway as the downstream signaling mechanism of BEST3. BEST3 deletion in SMCs suppressed MEKK2/3 ubiquitination, resulting in MEKK2/3 upregulation and MAPK phosphorylation, as evidenced by in vitro experiments. Genetic inhibition of MEKK2/3 using short hairpin RNA mitigated AngII-induced aortic rupture and aneurysm formation in SMC-BEST3 deficient mice. Ponatinib, a tyrosine-kinase inhibitor, also suppressed aortic rupture and aneurysm formation in AngII-infused SMC-BEST3−/− mice, along with the downregulation of MEKK2/3. Both the genetic and pharmacological approaches to MEKK2/3 inhibition also attenuated in aortopathies in apolipoprotein E deficient mice infused with AngII. Finally, the authors demonstrated that increased MEKK2/3 protein in human samples retrieved during repair of aortic dissection.

There is compelling evidence that MAPKs exert an important role in the pathophysiology of aortic diseases, including aortic aneurysms and dissection. ERK, JNK, and p38 are major members of the MAPK family, and many studies have shown increased phosphorylation of these molecules in aortopathies in humans and mice. In addition, inhibition of ERK, JNK, or their upstream components has been reported to suppress aortic dilatations in several types of aneurysm mouse models (Table 1).^5–9 These suggest that MAPK signaling pathways are key contributors to provoking aortic diseases. In the study by Zhang, et al,¹ BEST3 deletion in SMCs resulted in increased phosphorylation of ERK, JNK, and p38 that was associated with aortic dilatations and rupture. Interestingly, the inhibition of MEKK2/3, an upstream molecule of MAPKs, uniformly decreased MAPK activities and prevented AngII-mediated aortopathies. The observed prevention of aortopathies was not only seen in SMC-BEST3−/− mice but also observed in AngII-infused apolipoprotein E deficient mice, inferring that BEST3 was not an exclusive regulator of this pathway. These findings emphasize the significance of MEKK2/3 as a key driver in aortopathies. Thus, MEKK2/3 holds promise as a potential therapeutic target for aortic diseases, although more information is needed on the mechanistic basis for the involvement of this pathway.

Table 1.

Preclinical studies using MAPK or tyrosine kinase inhibitions for aortopathies.

Intervention	Model	Phenotype	Reference
MAPK Inhibition
MEKK2/3 silencing	AngII in SMC-BEST3−/−	AD, TAA, AAA ↓	Zhang, et al1
	AngII in ApoE−/−	AD, TAA, AAA ↓
MEK inhibitor (RDEA119, Trametinib)	Fbn1C1041G/+	TAA ↓	Holm, et al7
	Fbn1mgR/mgR with lactating	AD ↓	Habashi, et al8
	Col3a1G209S/+	AD, Aortic rupture ↓	Bowen, et al9
ERK1 deletion	Elastase	AAA ↓	Ghosh, et al5
JNK inhibitor (SP600125)	CaCl2	AAA ↓	Yoshimura, et al6
	AngII	AAA ↓
	Fbn1C1041G/+	TAA ↓	Holm, et al7
Tyrosine Kinase Inhibitors
Ponatinib	AngII in SMC-BEST3−/−	AD, TAA, AAA ↓	Zhang, et al1
	AngII in ApoE−/−	AD, TAA, AAA ↓
Imatinib	Elastase	AAA ↓	Yao, et al10
	AngII	AAA ↓	Vorkapic, et al11
Lenvatinib	Elastase	AAA ↓	Busch, et al12
Bosutinib	AngII	AAA ↔	Balbora Ramilo, et al13
Gefitinib	BAPN/AngII	AD ↔	Hayashi-Hori, et al14

AngII, indicate angiotensin II; ApoE−/−, apolipoprotein E deficient; AD, aortic dissection; AAA, abdominal aortic aneurysm TAA, thoracic aortic aneurysm; SMC, smooth muscle cell.

Zhang, et al¹ also demonstrated the prevention of aortopathies with the inhibition of MEKK2/3 in AngII-infused SMC-BEST3−/− mice by ponatinib, a multi-targeted tyrosine kinase inhibitor that is approved for use in patients with leukemia. There are many types of tyrosine kinase inhibitors and previous studies have tested the impact of several inhibitors on aortopathy formation in rodent models (Table 1). However, efficacy has been inconsistent among inhibitors. This is illustrated by imatinib and lenvatinib attenuating abdominal aortic aneurysms,^10–12 whereas bosutinib did not alter AngII-induced abdominal aortic aneurysms,¹³ and gefitinib did not change AngII/BAPN-induced aortic dissection.¹⁴ Although these drugs target different molecules in the tyrosine kinase pathway, a common feature is inhibition of MAPKs. Thus, these different outcomes in aortic pathologies highlight the need for further studies to define the role of tyrosine kinase-dependent MAPK activation in aortopathies. In addition, there are case reports showing complications of aortic dissections in patients using tyrosine kinase inhibitors. Thus, despite its approval for patients with leukemia, there are several impediments to the translation of ponatinib into clinical application for the prevention and treatment of aortopathies.

Aortic dissection is defined by the presence of a tear in the intima that results in a separation of medial layers and blood accumulating in the false lumen.¹⁵ Therefore, medial hematoma is a common characteristic of aortic dissections. This feature manifests in several aortopathy mouse models. This includes the administration of AngII or β-aminopropionitrile, either individually or simultaneously, and genetic manipulation models, such as SMC-specific inducible deletion of TGF-β receptor 1 or 2. Thus, medial hematoma is a hallmark characteristic for identifying aortic dissections in both humans and mice. In the study by Zhang, et al,¹ BEST3 deletion in SMCs led to significant aortic dilatation and spontaneous death due to aortic rupture, providing compelling evidence for a BEST3-linked MEKK2 pathway in maintaining aortic wall dimensions and integrity. However, further evidence is needed to determine whether medial dissection was a prelude to these aortopathies. Since medial hematomas can resolve, further study will be required that accounts for temporal progression in concert with detailed pathological tissue analysis.

In conclusion, the study by Zhang, et al¹ provides authoritative evidence of an important role of BEST3 regulation of MAPKs in SMCs in the integrity of the aortic wall. This study showed the potential therapeutic approach of focusing on MEKK2/3 inhibition, but there are several obstacles to extrapolating these data into clinical application.