CARASIL is associated with mutations in the HTRA1 gene. Our findings indicate a link between repressed inhibition of signaling by the TGF-beta family and ischemic cerebral small-vessel disease, alopecia, and spondylosis.

Cerebral small-vessel disease is a common disorder in elderly populations; however, its molecular basis is not well understood. We recently demonstrated that mutations in the high-temperature requirement A (HTRA) serine peptidase 1 (HTRA1) gene cause a hereditary cerebral small-vessel disease, cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). HTRA1 belongs to the HTRA protein family, whose members have dual activities as chaperones and serine proteases and also repress transforming growth factor-β (TGF-β) family signaling. We demonstrated that CARASIL-associated mutant HTRA1s decrease protease activity and fail to decrease TGF-β family signaling. However, the precise molecular mechanism for decreasing the signaling remains unknown. Here we show that increased expression of ED-A fibronectin is limited to cerebral small arteries and is not observed in coronary, renal arterial or aortic walls in patients with CARASIL. Using a cell-mixing assay, we found that HTRA1 decreases TGF-β1 signaling triggered by proTGF-β1 in the intracellular space. HTRA1 binds and cleaves the pro-domain of proTGF-β1 in the endoplasmic reticulum (ER), and cleaved proTGF-β1 is degraded by ER-associated degradation. Consequently, the amount of mature TGF-β1 is reduced. These results establish a novel mechanism for regulating the amount of TGF-β1, specifically, the intracellular cleavage of proTGF-β1 in the ER.

Endothelin 1 (ET-1), mainly produced from vascular endothelial cells, induces vasoconstriction in physiological conditions. The endothelin receptor antagonist is among the most effective agents for pulmonary hypertension. However, little is known about the production source of ET-1 in inflammation and immunity. Here, we studied whether T cell-mediated ET-1 production system exists and operates independent of the production system in vascular endothelial cells. ET-1 production was readily detectable in the culture supernatant of human PBMCs and murine spleen cells stimulated with anti-CD3 antibody. Immunocytostaining showed that ET-1-producing cells emerged only in PBMCs stimulated with anti-CD3 antibody. Using the Transwell system, both murine and human monocytes sorted with magnetic beads in the inner chamber produced ET-1 when T cells were activated with antigen or anti-CD3 antibody in the outer chamber. This ET-1 production was inhibited by anti-IFN-γ and/or TNF-α antibody. Furthermore, monocytes purified from ETflox/flox;Tie2-Cre( + ) mice, which conditionally lack ET-1 in hematopoietic stem cells and vascular endothelial cells, did not produce ET-1 even when stimulated by antigen-specific T cell activation. This study demonstrates the existence of an immune-mediated ET-1 production induced by T cells upon activation through IFN-γ and TNF-α.

Recent studies have shown that stem cell memory T (T SCM ) cell-like properties are important for successful adoptive immunotherapy by the chimeric antigen receptor-engineered-T (CAR-T) cells. We previously reported that both human and murineactivated T cells are converted into stem cell memory-like T (iT SCM ) cells by coculture with stromal OP9 cells expressing the NOTCH ligand. However, the mechanism of NOTCHmediated iT SCM reprogramming remains to be elucidated. Here, we report that the NOTCH/OP9 system efficiently converted conventional human CAR-T cells into T SCM -like CAR-T, "CAR-iT SCM " cells, and that mitochondrial metabolic reprogramming played a key role in this conversion. NOTCH signaling promoted mitochondrial biogenesis and fatty acid synthesis during iT SCM formation, which are essential for the properties of iT SCM cells. Forkhead box M1 (FOXM1) was identified as a downstream target of NOTCH, which was responsible for these metabolic changes and the subsequent iT SCM differentiation. Like NOTCH-induced CAR-iT SCM cells, FOXM1-induced CAR-iT SCM cells possessed superior antitumor potential compared with conventional CAR-T cells. We propose that NOTCHor FOXM1-driven CAR-iT SCM formation is an effective strategy for improving cancer immunotherapy.Significance: Manipulation of signaling and metabolic pathways important for directing production of stem cell memory-like T cells may enable development of improved CAR-T cells.

The results of this study demonstrated the reliability and validity of the PBQ and the 14-item version of the PBQ in Japanese mothers 4 weeks after delivery.