Maho Yamashita

Smooth Muscle–Selective Inhibition of Nuclear Factor-κB Attenuates Smooth Muscle Phenotypic Switching and Neointima Formation Following Vascular Injury

BACKGROUND Vascular proliferative diseases such as atherosclerosis are inflammatory disorders involving multiple cell types including macrophages, lymphocytes, endothelial cells, and smooth muscle cells (SMCs). Although activation of the nuclear factor-κB (NF-κB) pathway in vessels has been shown to be critical for the progression of vascular diseases, the cell-autonomous role of NF-κB within SMCs has not been fully understood. METHODS AND RESULTS We generated SMC-selective truncated IκB expressing (SM22α-Cre/IκBΔN) mice, in which NF-κB was inhibited selectively in SMCs, and analyzed their phenotype following carotid injury. Results showed that neointima formation was markedly reduced in SM22α-Cre/IκBΔN mice after injury. Although vascular injury induced downregulation of expression of SMC differentiation markers and myocardin, a potent activator of SMC differentiation markers, repression of these markers and myocardin was attenuated in SM22α-Cre/IκBΔN mice. Consistent with these findings, NF-κB activation by interleukin-1β (IL-1β) decreased expression of SMC differentiation markers as well as myocardin in cultured SMCs. Inhibition of NF-κB signaling by BAY 11-7082 attenuated repressive effects of IL-1β. Of interest, Krüppel-like factor 4 (Klf4), a transcription factor critical for regulating SMC differentiation and proliferation, was also involved in IL-1β-mediated myocardin repression. Promoter analyses and chromatin immunoprecipitation assays revealed that NF-κB repressed myocardin by binding to the myocardin promoter region in concert with Klf4. CONCLUSIONS These results provide novel evidence that activation of the NF-κB pathway cell-autonomously mediates SMC phenotypic switching and contributes to neointima formation following vascular injury.

Kruppel-like factor 4 contributes to high phosphate-induced phenotypic switching of vascular smooth muscle cells into osteogenic cells

Background: Elucidation of molecular mechanisms controlling vascular calcification is critical for chronic kidney disease patients. Results: High phosphate induced Klf4 expression in SMCs. Klf4 knockdown attenuated high phosphate-induced SMC phenotypic switching into osteogenic cells. Conclusion: Results suggest that Klf4 contributes to high phosphate-induced conversion of SMCs into osteogenic cells. Significance: Control of Klf4 might be a novel therapeutic target for vascular calcification. Hyperphosphatemia in chronic kidney disease is highly associated with vascular calcification. Previous studies have shown that high phosphate-induced phenotypic switching of vascular smooth muscle cells (SMCs) into osteogenic cells plays an important role in the calcification process. In the present study, we determined whether Krüppel-like factor 4 (Klf4) and phosphorylated Elk-1, transcriptional repressors of SMC differentiation marker genes activated by intimal atherogenic stimuli, contributed to this process. Rat aortic SMCs were cultured in the medium with normal (0.9 mmol/liter) or high (4.5 mmol/liter) phosphate concentration. Results showed that high phosphate concentration induced SMC calcification. Moreover, high phosphate decreased expression of SMC differentiation marker genes including smooth muscle α-actin and SM22α, whereas it increased expression of osteogenic genes, such as Runx2 and osteopontin. High phosphate also induced Klf4 expression, although it did not phosphorylate Elk-1. In response to high phosphate, Klf4 selectively bound to the promoter regions of SMC differentiation marker genes. Of importance, siRNA-mediated knockdown of Klf4 blunted high phosphate-induced suppression of SMC differentiation marker genes, as well as increases in expression of osteogenic genes and calcium deposition. Klf4 was also induced markedly in the calcified aorta of adenine-induced uremic rats. Results provide novel evidence that Klf4 mediates high phosphate-induced conversion of SMCs into osteogenic cells.

Kruppel-like Factor 4 Protein Regulates Isoproterenol-induced Cardiac Hypertrophy by Modulating Myocardin Expression and Activity

Background: The identification of novel molecular mechanisms for cardiac hypertrophy is of considerable interest. Results: Cardiomyocyte-specific knockout of KLF4 enhanced β-adrenoreceptor agonist-induced cardiac hypertrophy by modulating myocardin expression and activity. Conclusion: KLF4 is a novel regulator of cardiac hypertrophy. Significance: Control of KLF4 is a potential therapeutic target for cardiac hypertrophy. Kruppel-like factor 4 (KLF4) plays an important role in vascular diseases, including atherosclerosis and vascular injury. Although KLF4 is expressed in the heart in addition to vascular cells, the role of KLF4 in cardiac disease has not been fully determined. The goals of this study were to investigate the role of KLF4 in cardiac hypertrophy and to determine the underlying mechanisms. Cardiomyocyte-specific Klf4 knockout (CM Klf4 KO) mice were generated by the Cre/LoxP technique. Cardiac hypertrophy was induced by chronic infusion of the β-adrenoreceptor agonist isoproterenol (ISO). Results showed that ISO-induced cardiac hypertrophy was enhanced in CM Klf4 KO mice compared with control mice. Accelerated cardiac hypertrophy in CM Klf4 KO mice was accompanied by the augmented cellular enlargement of cardiomyocytes as well as the exaggerated expression of fetal cardiac genes, including atrial natriuretic factor (Nppa). Additionally, induction of myocardin, a transcriptional cofactor regulating fetal cardiac genes, was enhanced in CM Klf4 KO mice. Interestingly, KLF4 regulated Nppa expression by modulating the expression and activity of myocardin, providing a mechanical basis for accelerated cardiac hypertrophy in CM Klf4 KO mice. Moreover, we showed that KLF4 mediated the antihypertrophic effect of trichostatin A, a histone deacetylase inhibitor, because ISO-induced cardiac hypertrophy in CM Klf4 KO mice was attenuated by olmesartan, an angiotensin II type 1 antagonist, but not by trichostatin A. These results provide novel evidence that KLF4 is a regulator of cardiac hypertrophy by modulating the expression and the activity of myocardin.

Deletion of Krüppel-Like Factor 4 in Endothelial and Hematopoietic Cells Enhances Neointimal Formation Following Vascular Injury

Background Krüppel‐like factor 4 (Klf4) is involved in a variety of cellular functions by activating or repressing the transcription of multiple genes. Results of previous studies showed that tamoxifen‐inducible global deletion of the Klf4 gene in mice accelerated neointimal formation following vascular injury, in part via enhanced proliferation of smooth muscle cells (SMCs). Because Klf4 is also expressed in non‐SMCs including endothelial cells (ECs), we determined if Tie2 promoter‐dependent deletion of Klf4 in ECs and hematopoietic cells affected injury‐induced neointimal formation. Methods and Results Klf4 conditional knockout (cKO) mice were generated by breeding Tie2‐Cre mice and Klf4 floxed mice, and their phenotype was analyzed after carotid ligation injury. Results showed that injury‐induced repression of SMC differentiation markers was unaffected by Tie2 promoter‐dependent Klf4 deletion. However, of interest, neointimal formation was significantly enhanced in Klf4‐cKO mice 21 days following carotid injury. Moreover, Klf4‐cKO mice exhibited an augmented proliferation rate, enhanced accumulation of macrophages and T lymphocytes, and elevated expression of cell adhesion molecules including vascular cell adhesion molecule–1 (Vcam1) and E‐selectin in injured arteries. Mechanistic analyses in cultured ECs revealed that Klf4 inhibited tumor necrosis factor‐α–induced expression of Vcam1 through blocking the binding of nuclear factor‐κB to the Vcam1 promoter. Conclusions These results provide evidence that Klf4 in non‐SMCs such as ECs regulates neointimal formation by repressing arterial inflammation following vascular injury.

Endothelial krüppel-like factor 4 mediates the protective effect of statins against ischemic AKI

Endothelial cells participate in the pathophysiology of ischemic AKI by increasing the expression of cell adhesion molecules and by recruiting inflammatory cells. We previously showed that endothelial Krüppel-like factor 4 (Klf4) regulates vascular cell adhesion molecule 1 (Vcam1) expression and neointimal formation after carotid injury. In this study, we determined whether endothelial Klf4 is involved in ischemic AKI using endothelial Klf4 conditional knockout (Klf4 cKO) mice generated by breeding Tek-Cre mice and Klf4 floxed mice. Klf4 cKO mice were phenotypically normal before surgery. However, after renal ischemia-reperfusion injury, Klf4 cKO mice exhibited elevated serum levels of urea nitrogen and creatinine and aggravated renal histology compared with those of Klf4 floxed controls. Moreover, Klf4 cKO mice exhibited enhanced accumulation of neutrophils and lymphocytes and elevated expression of cell adhesion molecules, including Vcam1 and Icam1, in injured kidneys. Notably, statins ameliorated renal ischemia-reperfusion injury in control mice but not in Klf4 cKO mice. Mechanistic analyses in cultured endothelial cells revealed that statins increased KLF4 expression and that KLF4 mediated the suppressive effect of statins on TNF-α-induced VCAM1 expression by reducing NF-κB binding to the VCAM1 promoter. These results provide evidence that endothelial Klf4 is renoprotective and mediates statin-induced protection against ischemic AKI by regulating the expression of cell adhesion molecules and concomitant recruitment of inflammatory cells.

Inhalation of Hydrogen Gas Is Beneficial for Preventing Contrast-Induced Acute Kidney Injury in Rats.

Background: The present study aimed at investigating the effect of a novel antioxidant, hydrogen (H2) gas, on the severity of contrast-induced acute kidney injury (CIAKI) in a rat model. Methods: CIAKI was induced in rats by intravenous injection of a contrast medium, Ioversol, in addition to reagents inhibiting prostaglandin and nitric oxide synthesis. During the injection of these reagents, the rats inhaled H2 gas or control gas. Results: One day after the injection, serum levels of urea nitrogen were significantly lower in H2 gas-inhaling CIAKI rats (17.6 ± 2.3 mg/dl) than those in control gas-treated CIAKI rats (36.0 ± 7.3 mg/dl), although they both were elevated as compared to untreated rats (14.9 ± 0.9 mg/dl). Consistently, creatinine clearance in H2 gas-treated CIAKI rats was higher than that in control gas-treated counterparts. Renal histological analysis revealed that the formation of proteinaceous casts and tubular necrosis was improved by H2 gas inhalation. Mechanistic analyses showed that inhalation of H2 gas significantly reduced renal cell apoptosis, expression of cleaved caspase 3, and expression of an oxidative stress marker, 8-hydroxydeoxyguanosine, in injured kidneys. Conclusion: Results suggest that H2 gas inhalation is effective in ameliorating the severity of CIAKI in rats by reducing renal cell apoptosis and oxidative stress.

Generation of kidney tubular organoids from human pluripotent stem cells

Recent advances in stem cell research have resulted in methods to generate kidney organoids from human pluripotent stem cells (hPSCs), which contain cells of multiple lineages including nephron epithelial cells. Methods to purify specific types of cells from differentiated hPSCs, however, have not been established well. For bioengineering, cell transplantation, and disease modeling, it would be useful to establish those methods to obtain pure populations of specific types of kidney cells. Here, we report a simple two-step differentiation protocol to generate kidney tubular organoids from hPSCs with direct purification of KSP (kidney specific protein)-positive cells using anti-KSP antibody. We first differentiated hPSCs into mesoderm cells using a glycogen synthase kinase-3β inhibitor for 3 days, then cultured cells in renal epithelial growth medium to induce KSP+ cells. We purified KSP+ cells using flow cytometry with anti-KSP antibody, which exhibited characteristics of all segments of kidney tubular cells and cultured KSP+ cells in 3D Matrigel, which formed tubular organoids in vitro. The formation of tubular organoids by KSP+ cells induced the acquisition of functional kidney tubules. KSP+ cells also allowed for the generation of chimeric kidney cultures in which human cells self-assembled into 3D tubular structures in combination with mouse embryonic kidney cells.

High Glucose Concentration Does Not Modulate the Formation of Arterial Medial Calcification in Experimental Uremic Rats

High phosphate-induced phenotypic switching of smooth muscle cells (SMCs) into osteogenic cells is critical for the formation of arterial medial calcification in chronic kidney disease. Because vascular calcification is also prevalent in type 2 diabetes, we examined whether glucose concentration affects high phosphate-induced SMC phenotypic switching and calcification. First, the formation of arterial medial calcification was compared among 4 groups: adenine-fed uremic rats, streptozotocin-injected hyperglycemic rats, adenine-fed and streptozotocin-injected uremic/hyperglycemic rats, and control rats. Calcification was obvious in uremic and uremic/hyperglycemic rats, whereas it was undetectable in the others. Aortic calcium contents were significantly elevated in uremic and uremic/hyperglycemic rats, but they were not different between the two groups. Moreover, hyperglycemia had no effects on the reduced expression of SMC differentiation markers including smooth muscle α-actin and SM22α and on the increased expression of osteogenic markers, such as Runx2, in uremic rats. Second, cultured SMCs were incubated in the medium with various concentrations of phosphate (0.9-4.5 mmol/l) and glucose (5-50 mmol/l), and calcium deposition was measured. Although high phosphate dose-dependently increased calcium contents, they were unaffected by glucose concentration. Results suggest that glucose concentration does not directly modulate high phosphate-induced SMC phenotypic switching and arterial medial calcification.

Klotho protein supplementation reduces blood pressure and renal hypertrophy in db/db mice, a model of type 2 diabetes

Klotho interacts with various membrane proteins, such as receptors for transforming growth factor (TGF)‐β and insulin‐like growth factor (IGF), to alter their function. Renal expression of klotho is diminished in diabetes. The present study examined whether exogenous klotho protein supplementation ameliorates kidney injury and renin–angiotensin system (RAS) in db/db mice.

Smooth Muscle–Selective Nuclear Factor‐κB Inhibition Reduces Phosphate‐Induced Arterial Medial Calcification in Mice With Chronic Kidney Disease

Background Hyperphosphatemia is a major factor promoting the formation of arterial medial calcification in chronic kidney disease (CKD). However, arterial medial calcification begins to occur during the early stages of CKD, when hyperphosphatemia is not yet apparent. It is predicted that other factors also play a role. The aim of the present study was to determine the role of pro‐inflammatory nuclear factor‐κB (NF‐κB) signaling in smooth muscle cells (SMCs) for phosphate‐induced arterial medial calcification in CKD mice. Methods and Results We first sought to establish a novel mouse model of CKD with arterial medial calcification. CKD was induced in DBA/2 mice by feeding them a low concentration of adenine, and these mice were fed a normal or high‐phosphorus diet. Severe calcification was seen in CKD mice fed the high‐phosphorus diet, while it was undetectable in CKD mice fed the normal phosphorus diet or control mice fed the high‐phosphorus diet. Arterial medial calcification was accompanied by phenotypic switching of SMCs into osteogenic cells. Interestingly, NF‐κB inhibitors, tempol and triptolide, both reduced arterial medial calcification in CKD mice fed the high‐phosphorus diet. Moreover, formation of arterial medial calcification, as well as SMC phenotypic switching, was also markedly attenuated in transgenic mice, in which the NF‐κB activity was inhibited selectively in SMCs. Mechanistic studies revealed that Krüppel‐like factor 4 was involved in NF‐κB‐induced SMC phenotypic switching and calcification. Conclusions Results of the present studies suggest that the NF‐κB signaling in SMCs plays an important role in high phosphate‐induced arterial medial calcification in CKD.