Biochemical, genetic, and clinical evidence indicates that SMC proliferation in small pulmonary vessels is an essential part of the pathogenesis of pulmonary hypertension. This study identifies Notch3 as a crucial mediator of proliferation of sPASMCs and a crucial mediator for development of rodent PH, and possibly human PAH.
Our goal has been to understand the molecular basis by which normal pulmonary vessels develop smooth muscle hyperplasia and medial thickening which eventually occlude the distal pulmonary arterial tree and cause clinical manifestations of PAH. As a result of the work reported, we have four major conclusions. First, human PAH vasculopathy is characterized by high steady-state levels of NOTCH3 and its downstream effector, HES5, in SMCs lining small pulmonary arteries/arterioles. Our results establish a link between Notch3 signaling and the magnitude of PH in humans and animals. We demonstrate that the level of Notch3 protein is a sensitive molecular marker of severity of PAH in humans and PH in rodents. Second, constitutive Notch3 ICD expression induces sPASMC proliferation. This notion, coupled with the finding that NOTCH3 is overexpressed at mRNA and protein levels in the lungs of humans with PAH, support a potentially critical role of NOTCH signaling in mediating vSMC proliferation seen in this disease. To our knowledge, these findings are the first description of NOTCH/HES5 signaling in the adult lung vasculature. Our results establish a link between NOTCH3 signaling and the coordinate regulation of HES5 effector expression in the context of vSMC proliferation. We found that siRNA inhibition of HES5 expression causes a decrease in sPASMC proliferation and a shift in gene expression in vSMCs toward a more differentiated phenotype. Third, we show that Notch3 is requisite for the development of hypoxic PH in rodents. Notch3−/− mice are resistant to development of PH and are unable to generate a medial hypertrophic response to hypoxia, because Notch3-mediated proliferative and possibly antiapoptotic effect on sPASMC is required for the development of pulmonary vascular medial hypertrophy. Finally, we demonstrate that rodent PH can be effectively treated by blocking Notch3 signaling. Collectively, these results suggest that Notch3 signaling is required for the clinical and pathologic development of PAH/PH.
In the early stages of human PAH, the disease has two components: pulmonary vasoconstriction and vascular remodeling. As the disease progresses, the capacity of the pulmonary vascular bed to dilate and recruit unused vasculature is lost22–25
. Recently, Chantemele et al.19
suggested that Notch3 deficiency causes reduction in pressure-induced myogenic tone and enhanced flow-mediated dilation in tail artery of the rat, which is associated with reduction in RhoA activity. Broughton et al.26
has found that chronic-hypoxic rats have increased myogenic tone in small pulmonary arteries through a ROK-dependent myofilament Ca2+
sensitization. These results raise the possibility that Notch3 may modulate not just pulmonary vessel wall remodeling, but may also influence pulmonary vascular tone and dilation. Our results examining Notch3 in the pulmonary circulation have revealed little difference between Notch3+/+
mice with regards to response to vasoconstrictor drugs, flow-induced changes in pressure, and response to pulmonary vasodilators. Collectively, these results suggest that Notch3 may play different roles in the pulmonary and systemic circulations.
Clues as to why NOTCH3 may play a role in PAH come from studies of Notch function in modulating vSMC phenotype, its involvement with bone morphogenetic protein (BMP) signaling, and its role in the vessel wall in the setting of hypoxia. Notch-Hes/Hrt signaling has been associated with lack of cell cycle arrest in aortic SMCs9,13
, has been reported to repress myocardin-induced differentiation of myofibroblasts8
, and has been found to regulate SMC proliferation in neointimal injury in Hrt2-deficient and Notch1+/−
. Although these studies suggest a role of Notch signaling in SMC proliferation-homeostasis, none have been done in sPASMCs. One of the paradigms of Notch signaling is the observation that the biologic response to receptor activation is dependent on dosage as well as cellular and organ context29
. It is difficult to extend findings from different vascular beds or immortalized cell lines to the behavior of SMCs from the distal pulmonary vascular tree.
Notch3 may affect sPASMC remodeling in PH due to crosstalk with the Bmp receptor (Bmpr) signaling pathway. BMPR2
mutations have been found to be associated with the development of a familial human PAH30
. However, 40% of familial PAH individuals do not harbor mutations in BMPR2
, and most non-familial cases lack association with BMPR2
. This suggests that alternate or convergent pathways to BMPR signaling may play a role in this disease. Recently, signal integration between Notch and Bmpr has been found in several organs and cell types. Activation of Bmp signaling has been found to lead to enhanced transcription of the Notch target gene Herp2
), through binding of the intracellular Bmp-mediated transcription factor, Smad1, with Notch ICD32–34
. Interestingly, Hrt1 was then found to efficiently bind to and induce degradation of Id1, a downstream effector of Bmp signaling. Protein-protein interactions between Id2 (in Bmpr/Id signaling) and Hes1 (in Notch signaling) have also been demonstrated in the chicken hindbrain to be key regulators of expression of genes involved in neurogenesis33
. These feedback loops provide evidence that Notch signaling, in the context of certain organs, may modulate downstream Bmpr signaling.
A second line of evidence suggesting that NOTCH signaling may be essential to the development of PAH is its role in hypoxia, a known environmental inducer of this disease. Hypoxia is known to promote the undifferentiated cell state in various stem cell and precursor populations35
. A recent report shows that hypoxia requires functional Notch signaling to maintain cells in an undifferentiated state36
. In a mechanism similar to the crosstalk between Bmpr and Notch signaling, hypoxia inducible factor1-α (Hif1-α), an intracellular mediator of oxygen sensing, has been found bind to Notch ICD and act as a co-enhancer to stimulate transcription of Notch responsive genes under hypoxic conditions. Thus, NOTCH ICD is at the convergence point of two different signaling mechanisms: hypoxic HIF1-α signaling and BMP signaling, both of which have implicated in the development of PAH.
In summary, we find that high steady-state levels of NOTCH3 are associated with the development of PAH in humans and that Notch3 expression is obligate for the development of PH in two experimental models of this disease. Our work demonstrates that constitutive NOTCH3 signaling induces pulmonary vSMCs into a proliferative phenotype and that pulmonary hypertensive vascular pathology in vivo can be prevented by treatment with a drug that blocks Notch signaling. It is possible that other Notch receptors may also play a role in the development of PH, and more specific inhibitors of Notch3 signaling will need to be tested in the treatment of this disease. Our results suggest that molecular targeting of the NOTCH3-HES5 axis in pulmonary vascular smooth muscle may be a novel strategy for treatment of PAH based on the genetic profile of the pulmonary vascular wall in this disease. Future inhibition of the expression or effect of NOTCH3 signaling in the adult pulmonary vasculature may be a useful strategy to prevent and treat PAH in humans.