Hojin Kang

Angiogenesis is initiated in response to a variety of external cues, including mechanical and biochemical stimuli; however, the underlying signaling mechanisms remain unclear. Here, we investigated the proangiogenic role of the endothelial mechanosensor Piezo1. Genetic deletion and pharmacological inhibition of Piezo1 reduced endothelial sprouting and lumen formation induced by wall shear stress and proangiogenic mediator sphingosine 1-phosphate, whereas Piezo1 activation by selective Piezo1 activator Yoda1 enhanced sprouting angiogenesis. Similarly to wall shear stress, sphingosine 1-phosphate functioned by activating the Ca 2+ gating function of Piezo1, which in turn signaled the activation of the matrix metalloproteinase-2 and membrane type 1 matrix metalloproteinase during sprouting angiogenesis. Studies in mice in which Piezo1 was conditionally deleted in endothelial cells demonstrated the requisite role of sphingosine 1-phosphate-dependent activation of Piezo1 in mediating angiogenesis in vivo. These results taken together suggest that both mechanical and biochemical stimuli trigger Piezo1-mediated Ca 2+ influx and thereby activate matrix metalloproteinase-2 and membrane type 1 matrix metalloproteinase and synergistically facilitate sprouting angiogenesis.

The molecular mechanisms underlying the regulation of pluripotency by cellular metabolism in human embryonic stem cells (hESCs) are not fully understood. We found that high levels of glutamine metabolism are essential to prevent degradation of OCT4, a key transcription factor regulating hESC pluripotency. Glutamine withdrawal depletes the endogenous antioxidant glutathione (GSH), which results in the oxidation of OCT4 cysteine residues required for its DNA binding and enhanced OCT4 degradation. The emergence of the OCT4(lo) cell population following glutamine withdrawal did not result in greater propensity for cell death. Instead, glutamine withdrawal during vascular differentiation of hESCs generated cells with greater angiogenic capacity, thus indicating that modulating glutamine metabolism enhances the differentiation and functional maturation of cells. These findings demonstrate that the pluripotency transcription factor OCT4 can serve as a metabolic-redox sensor in hESCs and that metabolic cues can act in concert with growth factor signaling to orchestrate stem cell differentiation.

Endothelial cells are subjected to biochemical and mechanical stimuli, which regulate their angiogenic potential. We determined the synergistic effects of sphingosine-1-phosphate (S1P) and fluid wall shear stress (WSS) on a previously established model of human umbilical vein endothelial cell invasion into three-dimensional collagen matrices. Collagen matrices were incorporated into a parallel-plate flow chamber to apply controlled WSS to the surface of endothelial monolayers over a period of 24 h. Cell invasion required the presence of S1P, with the effects of S1P being enhanced by shear stress to an extent comparable with S1P combined with angiogenic growth factor stimulation. The number of invading cells depended on the magnitude of shear stress, with a maximal induction at a shear stress of approximately 5 dyn/cm2, whereas the invasion distance was proportional to the magnitude of shear stress. The enhancement of invasion by 5.3 dyn/cm2 shear stress coincided with elevated phosphorylation of Akt and matrix metalloproteinase (MMP)-2 activation. Furthermore, invasion induced by the combined application of WSS and S1P was attenuated by inhibitors of MMPs (GM6001) and the phosphatidylinositol 3-kinase/Akt signaling pathway (wortmannin). These results provide evidence that shear stress is a positive modulator of S1P-induced endothelial cell invasion into collagen matrices through enhanced Akt and MMP-2 activation.

Endothelial cells normally line the vasculature and remain quiescent. However, these cells can be rapidly stimulated to undergo morphogenesis and initiate new blood vessel formation given the proper cues. This study reports a new mechanism for initiating angiogenic sprout formation that involves vimentin, the major intermediate filament protein in endothelial cells. Initial studies confirmed vimentin was required for sphingosine 1-phosphate (S1P)- and growth factor (GF)-induced endothelial cell invasion, and vimentin was cleaved by calpains during invasion. Calpains were predominantly activated by GF and were required for sprout initiation. Because others have reported membrane type 1-matrix metalloproteinase (MT1-MMP) is required for endothelial sprouting responses, we tested whether vimentin and calpain acted upstream of MT1-MMP. Both calpain and vimentin were required for successful MT1-MMP membrane translocation, which was stimulated by S1P. In addition, vimentin complexed with MT1-MMP in a manner that required both the cytoplasmic domain of MT1-MMP and calpain activation, which increased the soluble pool of vimentin in endothelial cells. Altogether, these data indicate that pro-angiogenic signals converge to activate calpain-dependent vimentin cleavage and increase vimentin solubility, which act upstream to facilitate MT1-MMP membrane translocation, resulting in successful endothelial sprout formation in three-dimensional collagen matrices. These findings help explain why S1P and GF synergize to stimulate robust sprouting in 3D collagen matrices.

Abstract Disruption of alveolar–capillary barriers is a major complication of high-volume mechanical ventilation referred to as “ventilator-induced lung injury.” The stretching force in alveoli is transmitted to endothelial cells, increasing the tension on underlying endothelial plasma membrane. The mechanosensor Piezo1, a plasma membrane cation channel, was inducibly deleted in endothelial cells of mice (Piezo1iEC−/− ), which allowed us to study its role in regulating the endothelial barrier response to alveolar stretch. We observed significant increase in lung vascular permeability in Piezo1iEC−/− mice as compared with control Piezo1fl/fl mice in response to high-volume mechanical ventilation. We also observed that human lung endothelial monolayers depleted of Piezo1 and exposed to cyclic stretch had increased permeability. We identified the calcium-dependent cysteine protease calpain as a downstream target of Piezo1. Furthermore, we showed that calpain maintained stability of the endothelial barrier in response to mechanical stretch by cleaving Src kinase, which was responsible for disassembling endothelial adherens junctions. Pharmacological activation of calpain caused Src cleavage and thereby its inactivation, and it restored the disrupted lung endothelial barrier seen in Piezo1iEC−/− mice undergoing high-volume mechanical ventilation. Our data demonstrate that downregulation of Piezo1 signaling in endothelium is a critical factor in the pathogenesis of ventilator-induced lung injury, and thus augmenting Piezo1 expression or pharmacologically activating Piezo1 signaling may be an effective therapeutic strategy.