Rationale: B-cell leukemia 11b (BCL11B) is a transcription factor known as an essential regulator of T lymphocytes and neuronal development during embryogenesis. A genome-wide association study (GWAS) showed that a gene desert region downstream of BCL11B, known to function as a BCL11B enhancer, harbors single nucleotide polymorphisms (SNPs) associated with increased arterial stiffness. However, a role for BCL11B in the adult cardiovascular system is unknown. Objective: Based on these human findings, we sought to examine the relation between BCL11B and arterial function. Methods and Results: Here we report that BCL11B is expressed in the vascular smooth muscle (VSM) where it regulates vascular stiffness. RNA sequencing of aortas from WT and Bcl11b null mice (BSMKO) identified the cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) as the most significant differentially regulated signaling pathway in BSMKO compared to WT mice. BSMKO aortas showed decreased levels of PKG1, increased levels of Ca ++ -calmodulin-dependent serine/threonine phosphatase calcineurin (PP2B) and decreased levels of their common phosphorylation target, vasodilator-stimulated phosphoprotein (pVASP S239 ), a regulator of cytoskeletal actin rearrangements. Decreased pVASP S239 in BSMKO aortas was associated with increased actin polymerization (F/G actin ratio). Functionally, aortic force, stress, wall tension and stiffness, measured ex vivo in organ baths, were increased in BSMKO aortas, and BSMKO mice had increased pulse wave velocity, the in vivo index of arterial stiffness. Despite having no effect on blood pressure or microalbuminuria, increased arterial stiffness in BSMKO mice was associated with increased incidence of cerebral microbleeds compared to age-matched WT littermates. Conclusions: We have identified VSM BCL11B as a crucial regulator of aortic smooth muscle function and a potential therapeutic target for vascular stiffness.

BACKGROUNDArterial stiffness (AS) is the loss of compliance of elastic arteries known to increase the risk of cardiovascular events. A genome‐wide association study (GWAS) identified single nucleotide polymorphisms (SNPs) in a genetic locus downstream of BCL11B to be associated with increased AS, however a functional role for Bcl11b in the vasculature is unknown.OBJECTIVETo examine BCL11B's role in the aorta and vascular function including AS and blood pressure.METHODSTo study the role of BCL11B in the vasculature, we generated mice (SMMHCERCreT2‐Bcl11bfl/fl aka BSMKO) with tamoxifen‐inducible Bcl11b deletion in vascular smooth muscle (VSM). We measured pulse wave velocity (PWV), the gold standard in vivo measure of AS, by Doppler ultrasound, and blood pressure by telemetry in wild type (WT) and BSMKO mice. We measured isometric force, wall tension and stress in aortic rings of WT and BSMKO mice ex vivo in organ baths. We analyzed levels of filamentous (F) and globular (G) actin, smooth muscle myosin (MYH11), smooth muscle a‐actin (SMaA), phosphorylated and total VASP, and calcineurin by Western blot in WT and BSMKO VSM cell and aortic homogenates.RESULTSMice with Bcl11b deletion in VSM (BSMKO) exhibited higher PWV compared to WT littermates (3.1 ± 0.1 m/s in WT, n = 14 vs 3.8 ± 0.2 m/s in BSMKO, n = 17; p < 0.05), but had no detectable changes in blood pressure (systolic blood pressure, SBP: 121.8 ± 3.8 mmHg in WT, n = 7 vs 120.0 ± 2.3 mmHg in BSMKO, n = 8; NS). Compared to WT, BSMKO VSM cells had decreased mRNA and protein expression of VSM contractile proteins MYH11 (1.00 ± 0.05 A.U. in WT vs 0.42 ± 0.04 A.U. in BSMKO, n = 5 replicate experiments; p < 0.05) and SMA (1.00 ± 0.04 A.U. in WT vs 0.63 ± 0.06 A.U. in BSMKO, n = 4 replicate experiments; p < 0.05). Baseline force (1010 ± 96 mg in WT, n = 5 vs 1511 ± 106 mg in BSMKO, n = 7; p < 0.05), wall tension (1.72 ± 0.13 N/m in WT, n = 5 vs 2.60 ± 0.24 N/m in BSMKO, n = 7; p < 0.05) and stress (17.7 ± 1.5 kPa in WT, n = 5 vs 25.9 ± 2.2 kPa in BSMKO, n = 7; p < 0.05) generated by BSMKO aortic rings in organ baths were significantly increased compared to WT. In addition, BSMKO aortas exhibited increased F/G actin ratio indicating increased cytoskeletal actin polymerization (ratio of F to G actin: 4.82 ± 1.33 A.U. in WT, n = 6 vs 9.09 ± 1.02 A.U. in BSMKO, n = 7; p < 0.05). Phosphorylated VASP at S239, known to inhibit actin polymerization, was decreased (0.99 ± 0.04 A.U. in WT vs 0.19 ± 0.04 A.U. in BSMKO, n = 5 replicate experiments; p < 0.05), while PP2B, a phosphatase which regulates VASP phosphorylation, was increased in BSMKO aortas compared to WT. Treatment of BSMKO VSM cells with cyclosporine A (CSA, 10mM), an inhibitor of PP2B, reversed pVASP toward WT levels.CONCLUSIONSWe showed for the first time that BCL11B is present in VSM and regulates arterial stiffness at least in part by regulating contractile protein smooth muscle myosin and expression and cytoskeletal remodeling in VSM cells of the aorta via PP2B‐pVASP.Support or Funding InformationThis work was supported by NIH grant HL136311.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Background: B-cell leukemia 11b (Bcl11b) is a zinc-finger transcription factor known as master regulator of T lymphocytes and neuronal development during embryogenesis. However, a role for Bcl11b in the cardiovascular system has never been described. Based on human findings from a genome-wide association study (GWAS) that a gene desert region downstream of BCL11B , known to function as BCL11B enhancer, harbors single nucleotide polymorphisms (SNPs) associated with increased arterial stiffness, we sought to examine relations between Bcl11b and arterial function. Methods and Results: We found for the first time that Bcl11b is expressed in the vascular smooth muscle (VSM) of human and murine vasculature and transcriptionally regulates the expression of VSM contractile proteins smooth muscle myosin and smooth muscle α-actin. Lack of Bcl11b in VSM-specific Bcl11b null mice (BSMKO) resulted in an increased expression of Ca ++ -calmodulin-dependent serine/threonine phosphatase calcineurin in BSMKO VSM cells and aortas, which were inversely correlated with levels of phosphorylated VASP S239 , a calcineurin de-phosphorylation target. Decreased pVASP S239 in BSMKO aortas was associated with increased actin polymerization (F/G actin ratio), consistent with pVASP S239 ’s function as regulator of cytoskeletal actin rearrangements, and was normalized by treatment with calcineurin inhibitor cyclosporine A. Functionally, Bcl11b deletion in VSM cells translated in increased aortic force, stress and wall tension, measured ex vivo in BSMKO aortas in organ baths, and increased pulse wave velocity, the in vivo index of arterial stiffness, in BSMKO mice compared to WT littermates. Moreover, Bcl11b and pVASP S239 expression were decreased in aortas of obese and aged mice, two models of arterial stiffness. Bcl11b deletion in VSM had no effect on baseline blood pressure or angiotensin II-induced hypertension, measured in conscious WT and BSMKO mice by radiotelemetry, but dramatically increased the incidence of angII-induced aortic aneurysms in BSMKO mice. Conclusions: Taken together, our results identify VSM Bcl11b as a novel and crucial regulator of VSM cell phenotype and vascular structural and functional integrity