Tacrolimus rescues the signaling and gene expression signature of endothelial ALK1 loss-of-function and improves HHT vascular pathology

Abstract

Hereditary hemorrhagic telangiectasia (HHT) is a highly debilitating and life-threatening genetic vascular disorder arising from endothelial cell (EC) proliferation and hypervascularization, for which no cure exists. Because HHT is caused by loss-of-function mutations in bone morphogenetic protein 9 (BMP9)-ALK1-Smad1/5/8 signaling, interventions aimed at activating this pathway are of therapeutic value. We interrogated the whole-transcriptome in human umbilical vein ECs (HUVECs) and found that ALK1 signaling inhibition was associated with a specific pro-angiogenic gene expression signature, which included a significant elevation of DLL4 expression. By screening the NIH clinical collections of FDA-approved drugs, we identified tacrolimus (FK-506) as the most potent activator of ALK1 signaling in BMP9-challenged C2C12 reporter cells. In HUVECs, tacrolimus activated Smad1/5/8 and opposed the pro-angiogenic gene expression signature associated with ALK1 loss-of-function, by notably reducing Dll4 expression. In these cells, tacrolimus also inhibited Akt and p38 stimulation by vascular endothelial growth factor, a major driver of angiogenesis. In the BMP9/10-immunodepleted postnatal retina-a mouse model of HHT vascular pathology-tacrolimus activated endothelial Smad1/5/8 and prevented the Dll4 overexpression and hypervascularization associated with this model. Finally, tacrolimus stimulated Smad1/5/8 signaling in C2C12 cells expressing BMP9-unresponsive ALK1 HHT mutants and in HHT patient blood outgrowth ECs. Tacrolimus repurposing has therefore therapeutic potential in HHT.

Correcting Smad1/5/8, mTOR, and VEGFR2 treats pathology in hereditary hemorrhagic telangiectasia models

“…In the tBMP9/10ib retinas, Siro significantly reduced AVM number dosing, liquid chromatography/mass spectrometry (LC-MS) analyses measured average serum concentrations of 9.0 nM Siro and 5.3 nM Nin in the injected P6 pups. As shown in Figure 1A and described previously (22,32), vascular pathology in pups was initiated at P3 by 1 i.p. injection of the dams with anti-BMP9/10 antibodies.…”

“…In vitro data in cell cultures have further revealed that VEGFR2 phosphorylation and activity were increased upon ALK1 silencing (30), and that endoglin silencing affected VEG-FR2 trafficking to increase its downstream signaling (31). Transcriptomic data in ECs have also demonstrated that ALK1 negatively controlled VEGFR2 (KDR gene) expression (32,33). In vivo, Vegfr2 knockdown was reported to block hypervascularization and AVMs in Alk1 iΔEC mice (30) and pharmacological inhibition of VEGFR2 reduced disease severity in Eng iΔEC mice (31).…”

“…Recently, we reported the development of an HHT mouse model, which uses the transmammary route for administering BMP9-and BMP10-blocking antibodies to nursing mouse pups (22,32), hereafter referred to as the transmammary-treated, BMP9/10immunoblocked (tBMP9/10ib) mice. We showed that this model, which has the advantage of being less invasive for the pups and more suitable for the analysis of large cohorts, leads to reduced ALK1 signaling and the development of a robust HHT-like pathology in the retinal vasculature of the pups (22,32). In this model, we found that Tac significantly activated endothelial ALK1 signaling in vivo and prevented hypervascularization of the tBMP9/10ib retina.…”

“…In this model, we found that Tac significantly activated endothelial ALK1 signaling in vivo and prevented hypervascularization of the tBMP9/10ib retina. Further analyses of Tac efficacy in preventing vascular pathology in tBMP9/10ib mice revealed, however, a relatively modest effect of the drug on AVM development (32). These data prompted us to determine whether the macrolide analog of Tac, Siro (administered alone and in combination with Nin), is more potent in reducing the vascular pathology of the tBMP9/10ib retina.…”

“…and with Nin at P5 (0.3 mg/kg, i.p.). Mice were then analyzed at P6 ( Figure 1A), a time point at which retinal vessel dilation, hypervascularization, and AVMs can readily be observed and quantified in this model (22,32). In the tBMP9/10ib retinas, Siro significantly reduced AVM number dosing, liquid chromatography/mass spectrometry (LC-MS) analyses measured average serum concentrations of 9.0 nM Siro and 5.3 nM Nin in the injected P6 pups.…”