Abstract-Heat shock protein 72 (HSP72) is a stress-inducible protein capable of protecting a variety of cells from toxins, thermal stress, and ischemic injury. The cytoprotective role and mechanism of action of HSP72 in renal cell ischemic injury remain unclear. To study this, HSP72 was introduced (liposomal transfer) or induced (thermal stress, 43°Cϫ1 hour) in renal tubular cells (LLC-PK1) with Western blot confirmation. Cells were subjected to simulated ischemia 24 hours after liposomal HSP72 transfer or thermal stress, and the effect of HSP72 on nuclear factor-B (NF-B) activation (electrophoretic mobility shift assay and immunohistochemistry), IB␣ production (Western blot), postischemic tumor necrosis factor-␣ (TNF-␣) production (RT-PCR), and apoptosis (TUNEL assay) were determined. In separate experiments, the role of TNF-␣ in apoptosis was determined (anti-TNF-␣ neutralizing antibody). Results demonstrated that both liposomal transfer of HSP72 and thermal induction of HSP72 prevented NF-B activation and translocation, TNF-␣ gene transcription, and subsequent ischemia-induced renal tubular cell apoptosis. Furthermore, TNF-␣ neutralization also inhibited ischemia-induced renal tubular cell apoptosis. These results indicate that liposomal delivery of HSP72 inhibits ischemia-induced renal tubular cell apoptosis by preventing NF-B activation and subsequent TNF-␣ production. Further elucidation of the mechanisms of HSP-induced cytoprotection may result in therapeutic strategies that limit or prevent ischemia-induced renal damage.
Valvular and vascular calcification are common causes of cardiovascular morbidity and mortality. Developing effective treatments requires understanding the molecular underpinnings of these processes. Shear stress is thought to play a role in inhibiting calcification. Furthermore, NOTCH1 regulates vascular and valvular endothelium, and human mutations in NOTCH1 can cause calcific aortic valve disease. Here, we determined the genome-wide impact of altering shear stress and NOTCH signaling on aortic valve endothelium. mRNA-sequencing of human aortic valve endothelial cells (HAVECs) with or without knockdown of NOTCH1, in the presence or absence of shear stress, revealed NOTCH1-dependency of the atherosclerosis-related gene connexin 40 (GJA5), and numerous repressors of endochondral ossification. Among these, Matrix GLA Protein (MGP) is highly expressed in aortic valve and vasculature, and inhibits soft tissue calcification by sequestering bone morphogenetic proteins (BMPs). Altering NOTCH1 levels affected MGP mRNA and protein in HAVECs. Furthermore, shear stress activated NOTCH signaling and MGP in a NOTCH1-dependent manner. NOTCH1 positively regulated endothelial MGP in vivo through specific binding motifs upstream of MGP. Our studies suggest that shear stress activates NOTCH1 in primary human aortic valve endothelial cells leading to downregulation of osteoblast-like gene networks that play a role in tissue calcification.
Expression of heat shock proteins (HSP) is an adaptive response to cellular stress. Stress induces tumor necrosis factor (TNF)-alpha production. In turn, TNF-alpha induces HSP70 expression. However, osmotic stress or ultraviolet radiation activates TNF-alpha receptor I (TNFR-I) in the absence of TNF-alpha. We postulated that TNF-alpha receptors are involved in the induction of HSP70 by cellular stress. Peritoneal Mphi were isolated from wild-type (WT), TNF-alpha knockout (KO), and TNFR (I or II) KO mice. Cells were cultured overnight and then heat stressed at 43 +/- 0.5 degrees C for 30 min followed by a 4-h recovery at 37 degrees C. Cellular HSP70 expression was induced by heat stress or exposure to endotoxin [lipopolysaccharide (LPS)] as determined by immunoblotting. HSP70 expression induced by either heat or LPS was markedly decreased in TNFR-I KO Mphi, whereas TNFR-II KO Mphi exhibited HSP70 expression comparable to that in WT mice. Expression of HSP70 after heat stress in TNF-alpha KO Mphi was also similar to that in WT mice, suggesting that induction of HSP70 by TNFR-I occurs independently of TNF-alpha. In addition, levels of steady-state HSP70 mRNA were similar by RT-PCR in WT and TNFR-I KO Mphi despite differences in protein expression. Furthermore, the effect of TNFR-I appears to be cell specific, since HSP70 expression in splenocytes isolated from TNFR-I KO was similar to that in WT splenocytes. These studies demonstrate that TNFR-I is required for the synthesis of HSP70 in stressed Mphi by a TNF-independent mechanism and support an intracellular role for TNFR-I.
Background:
Atherosclerotic lesions preferentially occur at bifurcations and branching points where blood flow exerts low and bidirectional oscillatory shear stress (OSS) to the vascular walls. This study aims to determine the role of OSS in the induction of inflammatory activation and phenotypic transition in coronary artery endothelial cells and investigate the interaction between endothelial cells and smooth muscle cells in a coronary-artery-on-a-chip.
Methods and Results:
A three-dimensional microengineered human coronary-artery-on-a-chip was developed and incorporated with OSS to mimic the flow patterns within the microenvironment of coronary artery in atheroprone regions. Human coronary artery endothelial cells (HCAECs) were cultured on the upper surface of a collagen I-coated membrane, and human coronary artery smooth muscle cells (HCASMCs) were seeded on the opposite side of the membrane. Single-cell RNA sequencing analysis revealed that HCAECs were segregated into four subgroups after being exposed to OSS for 24 h, and inflammatory response and EndMT-related genes were enriched simultaneously in most HCAECs. OSS-induced inflammatory response and EndMT in HCAECs were confirmed by immunoblotting and immunofluorescence, and these changes were mediated by the Notch1/p38 MAPK-NF-κB signaling pathways. Moreover, HCAECs exposed to OSS induced extracellular matrix (ECM) protein remodeling and proliferation in HCASMCs through a paracrine mechanism. Multiplex ELISA analysis identified that RANTES exhibited the greatest increase after OSS in HCAEC culture and played a major role in modulation of ECM protein remodeling in HCASMCs. Further, IL-37 was able to prevent OSS-induced inflammatory response and EndMT in HCAECs and thereby abrogated ECM protein remodeling and proliferation in HCASMCs.
Conclusion:
OSS provokes inflammatory response and EndMT in HCAECs through activating the Notch1/p38 MAPK-NF-κB signaling pathways. The novel findings demonstrate that OSS-induced endothelial inflammatory response and associated EndMT promote vascular adverse remodeling and that anti-inflammatory cytokine IL-37 may have therapeutic potential for suppressing endothelial changes and resultant vascular adverse remodeling.
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