Objective: Pulmonary arterial hypertension is a disease of proliferative vascular occlusion that is strongly linked to mutations in BMPR2 —the gene encoding the BMPR-II (BMP [bone morphogenetic protein] type II receptor). The endothelial-selective BMPR-II ligand, BMP9, reverses disease in animal models of pulmonary arterial hypertension and suppresses the proliferation of healthy endothelial cells. However, the impact of BMPR2 loss on the antiproliferative actions of BMP9 has yet to be assessed. Approach and Results: BMP9 suppressed proliferation in blood outgrowth endothelial cells from healthy control subjects but increased proliferation in blood outgrowth endothelial cells from pulmonary arterial hypertension patients with BMPR2 mutations. This shift from growth suppression to enhanced proliferation was recapitulated in control human pulmonary artery endothelial cells following siRNA-mediated BMPR2 silencing, as well as in mouse pulmonary endothelial cells isolated from endothelial-conditional Bmpr2 knockout mice ( Bmpr2 EC −/ − ). BMP9-induced proliferation was not attributable to altered metabolic activity or elevated TGFβ (transforming growth factor beta) signaling but was linked to the prolonged induction of the canonical BMP target ID1 in the context of BMPR2 loss. In vivo, daily BMP9 administration to neonatal mice impaired both retinal and lung vascular patterning in control mice ( Bmpr2 EC+/+ ) but had no measurable effect on mice bearing a heterozygous endothelial Bmpr2 deletion ( Bmpr2 EC+/− ) and caused excessive angiogenesis in both vascular beds for Bmpr2 EC −/− mice. Conclusions: BMPR2 loss reverses the endothelial response to BMP9, causing enhanced proliferation. This finding has potential implications for the proposed translation of BMP9 as a treatment for pulmonary arterial hypertension and suggests the need for focused patient selection in clinical trials.

Angiogenic sprouting can contribute adaptively, or mal-adaptively, to a myriad of conditions including ischemic heart disease and cancer. While the cellular and molecular systems that regulate tip versus stalk endothelial cell (EC) specification during angiogenesis are known, those systems that regulate their distinct actions remain poorly understood. Pre-clinical and clinical findings support sustained adrenergic signaling in promoting angiogenesis, but links between adrenergic signaling and angiogenesis are lacking; importantly, adrenergic agents alter the activation status of the cAMP signaling system. Here, we show that the cAMP effector, PKA, acts in a cell autonomous fashion to constitutively reduce the in vitro and ex vivo angiogenic sprouting capacity of ECs. At a cellular level, we observed that silencing or inhibiting PKA in human ECs increased their invasive capacity, their generation of podosome rosettes and, consequently, their ability to degrade a collagen matrix. While inhibition of either Src-family kinases or of cdc42 reduced these events in control ECs, only cdc42 inhibition, or silencing, significantly impacted them in PKA(Cα)-silenced ECs. Consistent with these findings, cell-based measurements of cdc42 activity revealed that PKA activation inhibits EC cdc42 activity, at least in part, by promoting its interaction with the inhibitory regulator, guanine nucleotide dissociation inhibitor-α (RhoGDIα).

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In addition to maintaining cellular ER Ca 2+ stores, store-operated Ca 2+ entry (SOCE) regulates several Ca 2+ -sensitive cellular enzymes, including certain adenylyl cyclases (ADCYs), enzymes that synthesize the secondary messenger cyclic AMP (cAMP). Ca 2+ , acting with calmodulin, can also increase the activity of PDE1-family phosphodiesterases (PDEs), which cleave the phosphodiester bond of cAMP. Surprisingly, SOCE-regulated cAMP signaling has not been studied in cells expressing both Ca 2+ -sensitive enzymes. Here, we report that depletion of ER Ca 2+ activates PDE1C in human arterial smooth muscle cells (HASMCs). Inhibiting the activation of PDE1C reduced the magnitude of both SOCE and subsequent Ca 2+ /calmodulin–mediated activation of ADCY8 in these cells. Because inhibiting or silencing Ca 2+ -insensitive PDEs had no such effects, these data identify PDE1C-mediated hydrolysis of cAMP as a novel and important link between SOCE and its activation of ADCY8. Functionally, we showed that PDE1C regulated the formation of leading-edge protrusions in HASMCs, a critical early event in cell migration. Indeed, we found that PDE1C populated the tips of newly forming leading-edge protrusions in polarized HASMCs, and co-localized with ADCY8, the Ca 2+ release activated Ca 2+ channel subunit, Orai1, the cAMP-effector, protein kinase A, and an A-kinase anchoring protein, AKAP79. Because this polarization could allow PDE1C to control cAMP signaling in a hyper-localized manner, we suggest that PDE1C-selective therapeutic agents could offer increased spatial specificity in HASMCs over agents that regulate cAMP globally in cells. Similarly, such agents could also prove useful in regulating crosstalk between Ca 2+ /cAMP signaling in other cells in which dysregulated migration contributes to human pathology, including certain cancers.

While fast flowing blood in linear arterial segments exposes arterial vascular endothelial cells (VECs) to high levels of laminar fluid shear stress (FSS, 15–20 dyne/cm2), slow or oscillatory blood flow, such as is present at arterial bifurcations, exposes VECs to modest levels of non‐linear FSS (1–3 dyne/cm2). These hemodynamic features make arterial bifurcations, arches and branch points especially prone to atherosclerosis. Exposing VECs to high laminar FSS promotes establishment of an adaptive (anti‐thrombotic, anti‐atherogenic and barrier forming) phenotype, an effect that is reversed following exposure of these cells to low / non‐linear levels of FSS. Previously we reported that a cAMP‐signalosome, composed of exchange protein activated by cAMP1 (EPAC1), Rap1 and a phosphodiesterase 4D (PDE4D) allowed cAMP to promote barrier functions in static VEC cultures. This signalosome was shown to associate with components of VEC adherens junctions, including VE‐cadherin (VECAD) and β‐catenin. Recently, we reported that activation of this same AJ‐associated signalosome allowed cAMP, via EPAC1‐mediated activation of Rap1, to promote expression of an adaptive phenotype even in VECs exposed to low levels of FSS. Indeed, siRNA‐mediated knockdown of EPAC1, Rap1 or PDE4D reduced the adaptive responses of VECs exposed to FSS and increased their pro‐thrombotic and pro‐angiogenic potential. Interestingly, PDE4D knockdown reduced responses in these cells to levels equivalent to those detected in cells in which EPAC1 was knocked down. We reported that this effect was due to the fact that PDE4D acts as a tether to localize EPAC1 within this important cAMP‐signalosome. Recently we identified phosphodiesterase 4D7 (PDE4D7) as the PDE4D variant that populates this VEC cAMP‐signalosome. Notably, VEC alignment in the direction of flow was reduced by knockdown of either pan‐PDE4D, selective PDE4D7 or EPAC1 when subjected to low FSS. Both Pan‐PDE4D or selective PDE4D7 siRNA‐mediated knockdown markedly reduced the ability of VECs to respond adaptively to FSS, and promoted loss of EPAC1 from the cAMP‐signalosome. In contrast, PDE4 inhibitors only modestly altered these responses in VECs exposed to various levels of FSS. These findings, together with our earlier work, are consistent with the idea that the tethering actions of PDE4D7 are dominant in this complex in VECs. Using telomerase‐immortalized human aortic endothelial cells, we created a cell‐based system in which the separate catalytic and tethering actions of PDE4D7 in controlling VEC responses to FSS could be measured. Overall, our findings are consistent with the novel idea that the EPAC1 tethering function of PDE4D7 is dominant in regulating EPAC1/Rap1‐mediated effects in VECs and that the cAMP hydrolytic function of PDE4D7 has a hyperlocalized impact within the AJ‐delimited nanodomain. Our findings pave the way for the development of displacing peptide (DP)‐based tools through which to regulate cAMP‐mediated function in VECs exposed to mal‐adaptive levels of FSS.Support or Funding InformationFunding provided by the CIHR.