CD47-mediated immune evasion in early-stage lung cancer progression.

Alveolar and interstitial macrophages play crucial roles in eradicating pathogens and transformed cells in the lungs. The immune checkpoint CD47, found on normal and malignant cells, interacts with the SIRPα ligand on macrophages, inhibiting phagocytosis, antigen presentation, and promoting immune evasion. In this study, we demonstrated that CD47 is not only a transmembrane protein, but that it is also highly concentrated in extracellular vesicles from lung cancer cell lines and patient plasma. Abundant CD47 was observed in the cytoplasm of lung cancer cells, aligning with our finding that it was packed into extracellular vesicles for physiological and pathological functions. In our clinical cohort, extracellular vesicle CD47 was significantly higher in the patients with early-stage lung cancer, emphasizing innate immunity inactivation in early tumor progression. To validate our hypothesis, we established an orthotopic xenograft model mimicking lung cancer development, which showed increased serum soluble CD47 and elevated IL-10/TNF-α ratio, indicating an immune-suppressive tumor microenvironment. CD47 expression led to reduced tumor-infiltrating macrophages during progression, while there was a post-xenograft increase in tumor-associated macrophages. In conclusion, CD47 is pivotal in early lung cancer progression, with soluble CD47 emerging as a key pathological effector.

Association Between Trimethylamine N-oxide and Adverse Kidney Outcomes and Overall Mortality in Type 2 Diabetes Mellitus.

CONTEXT: Type 2 diabetes (T2D) is the major contributor to chronic kidney disease and end-stage kidney disease (ESKD). The influence of trimethylamine N-oxide (TMAO) on kidney outcomes in T2D remains unclear. OBJECTIVE: To examine the association between fasting serum TMAO levels and adverse kidney outcomes in patients with T2D. METHODS: Between October 2016 and June 2020, patients with T2D were recruited and monitored every 3 months until December 2021. Serum TMAO levels were assessed using liquid chromatography-mass spectrometry. The primary kidney outcomes were doubling of serum creatinine levels or progression to ESKD necessitating dialysis; the secondary kidney outcome was a rapid 30% decline in estimated glomerular filtration rate within 2 years. All-cause mortality was also evaluated. RESULTS: Among the 440 enrolled patients with T2D, those in the highest serum TMAO tertile (≥0.88 µM) were older, had a longer diabetes duration, elevated blood urea nitrogen, and lower estimated glomerular filtration rate. Over a median follow-up period of 4 years, 26 patients (5.9%) had a doubling of serum creatinine level or progression to ESKD. After propensity score weighting, the patients in the highest serum TMAO tertile had a 6.45-fold increase in the risk of doubling of serum creatinine levels or progression to ESKD and 5.86-fold elevated risk of rapid decline in kidney function compared with those in the lowest tertile. Additionally, the stepwise increase in serum TMAO was associated with all-cause mortality. CONCLUSION: Patients with T2D with elevated circulating TMAO levels are at higher risk of doubling serum creatinine, progressing to ESKD, and mortality. TMAO is a potential biomarker for kidney function progression and mortality in patients with T2D.

Proteins Secreted by Lung Cancer Cells Induce the Onset of Proteinuria via Focal Adhesion Kinase Signaling in Mice.

Paraneoplastic nephrotic syndrome (PNS) is a complication seen in cancer patients. Ultrastructural examination shows the accumulation of proteins and the presence of foot process (FP) effacement in the glomeruli of PNS patients. Previously, we reported that orthotopic xenografts of Lewis lung carcinoma 1 in C57BL/6 mice caused them to develop lung cancer with albuminuria. This implies that these mice can be used as a model of human disease and suggests that Lewis lung carcinoma 1 cell-secreted proteins (LCSePs) contain nephrotoxic molecules and cause inflammation in renal cells. As podocyte effacement was present in glomeruli in this model, such podocyte injury may be attributable to either soluble LCSeP or LCSeP deposits triggering pathological progression. LCSePs in conditioned media was concentrated for nephrotoxicity testing. Integrin-focal adhesion kinase (FAK) signaling and inflammatory responses were evaluated in podocytes either exposed to soluble LCSePs or seeded onto substrates with immobilized LCSePs. FAK phosphorylation and interleukin-6 expression were higher in podocytes attached to LCSePs substrates than in those exposed to soluble LCSePs. Notably, LCSeP-based haptotaxis gave rise to altered signaling in podocytes. When podocytes were stimulated by immobilized LCSePs, FAK accumulated at focal adhesions, synaptopodin dissociated from F-actin, and disrupting the interactions between synaptopodin and α-actinin was observed. When FAK was inhibited by PF-573228 in immobilized LCSePs, the association between synaptopodin and α-actinin was observed in the podocytes. The association of synaptopodin and α-actinin with F-actin allowed FP stretching, establishing a functional glomerular filtration barrier. Therefore, in this mouse model of lung cancer, FAK signaling prompts podocyte FP effacement and proteinuria, indicative of PNS.

Coordination of LMO7 with FAK Signaling Sustains Epithelial Integrity in Renal Epithelia Exposed to Osmotic Pressure.

The kidney epithelial barrier has multifaceted functions in body fluids, electrolyte homeostasis, and urine production. The renal epithelial barrier (REB) frequently faces and challenges osmotic dynamics, which gives rise to osmotic pressure (a physical force). Osmotic pressure overloading can crack epithelial integrity and damage the REB. The endurance of REB to osmotic pressure forces remains obscure. LMO7 (LIM domain only 7) is a protein associated with the cell-cell junctional complex and cortical F-actin. Its upregulation was observed in cells cultured under hypertonic conditions. LMO7 is predominantly distributed in renal tubule epithelial cells. Hypertonic stimulation leads to LMO7 and F-actin assembly in the cortical stress fibers of renal epithelial cells. Hypertonic-isotonic alternation, as a pressure force pushing the plasma membrane inward/outward, was set as osmotic disturbance and was applied to test FAK signaling and LMO7 functioning in maintaining junctional integrity. LMO7 depletion in cells resulted in junctional integrity loss in the epithelial sheet-cultured hypertonic medium or hypertonic-isotonic alternation. Conversely, FAK inhibition by PF-573228 led to failure in robust cortical F-actin assembly and LMO7 association with cortical F-actin in epithelial cells responding to hypertonic stress. Epithelial integrity against osmotic stress and LMO7 and FAK signaling are involved in assembling robust cortical F-actin and maintaining junctional integrity. LMO7 elaborately manages FAK activation in renal epithelial cells, which was demonstrated excessive FAK activation present in LMO7 depleted NRK-52E cells and epithelial integrity loss when cells with LMO7 depletion were exposed to a hypertonic environment. Our data suggests that LMO7 regulates FAK activation and is responsible for maintaining REB under osmotic disturbance.

Predictive Value of HbA1c and Metabolic Syndrome for Renal Outcome in Non-Diabetic CKD Stage 1-4 Patients.

Glycated hemoglobin (HbA1c) levels are commonly used to indicate long-term glycemic control. An HbA1c level of 6.5−5.7% is defined as pre-diabetes and is proposed as a criterion for diagnosing metabolic syndrome (MetS). However, HbA1c levels can be affected by chronic kidney disease (CKD). Whether HbA1c is associated with clinical outcomes in nondiabetic CKD patients with or without MetS is still unknown. This study included 1270 nondiabetic CKD stage 1−4 Asian patients, divided by HbA1c and MetS. Through linear regression, HbA1c was positively associated with age, waist circumference, hemoglobin levels, and C-reactive protein and was negatively associated with malnutrition−inflammation. HbA1c levels were 5.5% (0.6%) and 5.7% (0.6%) in non-MetS and MetS, respectively (p < 0.001). In Cox regression, higher-level HbA1c was associated with worse composite renal outcome in MetS patients, but with better renal outcome in non-MetS patients: Hazard ratio (HR) (95% confidence interval [CI]) of HbA1c ≥5.7%, compared with HbA1c <5%, was 2.00 (1.06−3.78) in MetS and 0.25 (0.14−0.45) in non-MetS. An association between HbA1c and all-cause mortality was not found. In conclusion, higher HbA1c levels are associated with worse renal outcomes in nondiabetic CKD stage 1−4 patients modified by the presence of MetS.

FAK Executes Anti-Senescence via Regulating EZH2 Signaling in Non-Small Cell Lung Cancer Cells.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase overexpressed in various cancer types that plays a critical role in tumor progression. Accumulating evidence suggests that targeting FAK, either alone or in combination with other agents, may serve as an effective therapeutic strategy for numerous cancers. In addition to retarding proliferation, metastasis, and angiogenesis, FAK inhibition triggers cellular senescence in lung cancer cells. However, the detailed mechanism remains enigmatic. In the present study, we found that FAK inhibition not only elicits DNA-damage signaling but also downregulates enhancer of zeste homolog 2 (EZH2) expression. The manipulation of FAK expression influences EZH2 expression and corresponding signaling in vitro. Immunohistochemistry shows that active FAK signaling corresponds with the activation of the EZH2-mediated signaling cascade in lung-cancer-cells-derived tumor tissues. We also found that ectopic EZH2 expression attenuates FAK-inhibition-induced cellular senescence in lung cancer cells. Our results identify EZH2 as a critical downstream effector of the FAK-mediated anti-senescence pathway. Targeting FAK-EZH2 axis-induced cellular senescence may represent a promising therapeutic strategy for restraining tumor growth.

Hyperuricemia, a Non-Independent Component of Metabolic Syndrome, Only Predicts Renal Outcome in Chronic Kidney Disease Patients without Metabolic Syndrome or Diabetes.

Uric acid (UA) is elevated in metabolic syndrome (MS) and diabetes (DM). UA is associated with central obesity and blood glucose and is proposed as a criterion of MS. Previous reports showed that UA could predict renal outcome in CKD. However, recent clinical trials did not demonstrate the benefits of urate-lowering agents (ULA) for renal outcome. Whether the prognostic value of UA for renal outcome is independent of MS or secondary to MS in CKD patients is unknown. Our study included 2500 CKD stage 1−4 Asian patients divided by UA tertiles and MS/DM. In linear regression, UA was associated with obesity, C-reactive protein, and renal function. In Cox regression, high UA was associated with worse renal outcome in non-MS/DM, but not in MS/DM: hazard ratio (95% confidence interval) of UA tertile 3 was 3.86 (1.87−7.97) in non-MS/DM and 1.00 (0.77−1.30) in MS/DM (p for interaction < 0.05). MS was associated with worse renal outcome, but redefined MS (including hyperuricemia as the 6th criteria) was not. In conclusion, hyperuricemia is associated with worse renal outcome in non-MS/DM and is not an independent component of MS in CKD stage 1−4 patients. Hyperuricemia secondary to MS could not predict renal outcome.

FAK in Cancer: From Mechanisms to Therapeutic Strategies.

Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, is overexpressed and activated in many cancer types. FAK regulates diverse cellular processes, including growth factor signaling, cell cycle progression, cell survival, cell motility, angiogenesis, and the establishment of immunosuppressive tumor microenvironments through kinase-dependent and kinase-independent scaffolding functions in the cytoplasm and nucleus. Mounting evidence has indicated that targeting FAK, either alone or in combination with other agents, may represent a promising therapeutic strategy for various cancers. In this review, we summarize the mechanisms underlying FAK-mediated signaling networks during tumor development. We also summarize the recent progress of FAK-targeted small-molecule compounds for anticancer activity from preclinical and clinical evidence.

Targeting Pin1 for Modulation of Cell Motility and Cancer Therapy.

Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) specifically binds and isomerizes the phosphorylated serine/threonine-proline (pSer/Thr-Pro) motif, which leads to changes in protein conformation and function. Pin1 is widely overexpressed in cancers and plays an important role in tumorigenesis. Mounting evidence has revealed that targeting Pin1 is a potential therapeutic approach for various cancers by inhibiting cell proliferation, reducing metastasis, and maintaining genome stability. In this review, we summarize the underlying mechanisms of Pin1-mediated upregulation of oncogenes and downregulation of tumor suppressors in cancer development. Furthermore, we also discuss the multiple roles of Pin1 in cancer hallmarks and examine Pin1 as a desirable pharmaceutical target for cancer therapy. We also summarize the recent progress of Pin1-targeted small-molecule compounds for anticancer activity.

Lung Cancer Cell-Derived Secretome Mediates Paraneoplastic Inflammation and Fibrosis in Kidney in Mice.

Kidney failure is a possible but rare complication in lung cancer patients that may be caused by massive tumor lysis or a paraneoplastic effect. Clinical case reports have documented pathological characteristics of paraneoplastic syndrome in glomeruli, but are short of molecular details. When Lewis lung carcinoma 1 (LLC1) cells were implanted in mice lungs to establish lung cancer, renal failure was frequently observed two weeks post orthotopic xenograft. The high urinary albumin-to-creatinine ratio (ACR) was diagnosed as paraneoplastic nephrotic syndrome in those lung cancer mice. Profiling the secretome of the lung cancer cells revealed that the secretory proteins were potentially nephrotoxic. The nephrotoxicity of lung cancer-derived secretory proteins was tested by examining the pathogenic effects of 1 × 106, 2 × 106, and 5 × 106 LLC1 cell xenografts on the pathogenic progression in kidneys. Severe albuminuria was present in the mice that received 5 × 106 LLC1 cells implantation, whereas 106 cell and 2 × 106 cell-implanted mice have slightly increased albuminuria. Pathological examinations revealed that the glomeruli had capillary loop collapse, tumor antigen deposition in glomeruli, and renal intratubular casts. Since IL-6 and MCP-1 are pathologic markers of glomerulopathy, their distributions were examined in the kidneys of the lung cancer mice. Moderate to severe inflammation in the kidneys was correlated with increases in the number of cells implanted in the mice, which was reflected by renal IL-6 and MCP-1 levels, and urine ACR. TGF-ß signaling-engaged renal fibrosis was validated in the lung cancer mice. These results indicated that lung cancer cells could provoke inflammation and activate renal fibrosis.

Renal Damages in Deoxycorticosterone Acetate-Salt Hypertensive Rats: Assessment with Diffusion Tensor Imaging and T2-mapping.

PURPOSE: This study aimed to investigate the feasibility of diffusion tensor imaging (DTI) and T2-mapping to assess temporal renal damage in deoxycorticosterone acetate-salt (DOCA-salt) hypertensive rats and compare the results with histopathologic and immunohistochemical findings. PROCEDURES: After baseline renal magnetic resonance imaging (MRI), 24 out of 30 uninephrectomized Sprague-Dawley rats with DOCA-salt-induced hypertension were divided equally into four groups. Group 1 had renal MRI at weeks 2, 4, 6, and 8, and groups 2, 3, and 4 had MRI at weeks 2, 4, and 6, respectively. The remaining 6 rats were used as sham controls. The renal cortex and outer and inner stripes of the outer medulla were examined over time using fractional anisotropy (FA), apparent diffusion coefficient (ADC), and T2-mapping, and the results were compared with baseline values. The degree of glomerular and tubular injury, endothelial cell thickening, hyaline arteriolosclerosis, macrophage infiltration, microcyst formation, and fibrosis in different zones at different time points in the DOCA-salt rats were compared with controls. RESULTS: Compared with baseline values, DOCA-salt rats demonstrated a significant decrease in renal cortical FA from week 4 to week 8 (0.244 ± 0.015 vs 0.172 ± 0.014-0.150 ± 0.016, P = 0.018-0.002), corresponding to significantly more glomerular damage, arteriolosclerosis, macrophage infiltration, and fibrosis. The DOCA-salt rats had significantly increased cortical ADC and T2 values at weeks 6 and 8 (1.778 ± 0.051 × 10-3 mm2/s vs 1.872 ± 0.058-1.917 ± 0.066 × 10-3 mm2/s; 93.7 ± 4.9 ms vs 98.0 ± 2.9-100.7 ± 4.0 ms, respectively, all P < 0.05), consistent with excessively fluid-filled microcysts (aquaporin-2+). Despite DOCA-salt rats harbored markedly increased fibrosis in outer and inner stripes of the outer medulla at weeks 6 and 8, only nonsignificant decreases in FA were observed in comparison with the controls suggesting that only limited microstructural changes were present. CONCLUSIONS: Renal cortical FA is useful for the early detection and monitoring of renal damage in DOCA-salt hypertensive rats.

Predominant global glomerulosclerosis in patients of upper urinary tract urothelial carcinoma with pre-existing renal function impairment is a predictor of poor renal outcomes.

BACKGROUND: Incidence of renal dysfunction and risks of progression to end-stage renal disease (ESRD) were reported higher in upper urinary tract urothelial carcinoma (UTUC) than in renal cell carcinoma (RCC) patients after unilateral nephrectomy. METHODS: Totally 193 renal cancer patients, including 132 UTUC and 61 RCC, were studied to clarify whether the pathological changes of the kidney remnant removed from nephrectomy and the clinical factors might predict the risk of ESRD. Renal tubulointerstitial (TI) score and global glomerulosclerosis (GGS) rate were examined by one pathologist and two nephrologists independently under same histopathological criteria. RESULTS: The glomerular filtration rates at the time of surgery were lower in UTUC than RCC groups (p < 0.001). Average GGS score and average TI rate were higher in UTUC than in RCC groups (p < 0.001; p < 0.001). Competitive risk factor analysis revealed that abnormal GGS rate not related to age, predominant in UTUC with pre-existing renal function impairment, was a histopathological predictor of poor renal outcomes (creatinine doubling or ESRD) within 5 years in UTUC patients. CONCLUSION: Pre-existing renal function and pathological change of kidney remnant in both UTUC and RCC have the value for prediction of renal outcomes.

Inhibition of FAK Signaling Elicits Lamin A/C-Associated Nuclear Deformity and Cellular Senescence.

Focal adhesion kinase (FAK) is a non-receptor kinase that facilitates tumor aggressiveness. The effects of FAK inhibition include arresting proliferation, limiting metastasis, and inhibiting angiogenesis. PF-573228 is an ATP-competitive inhibitor of FAK. Treating lung cancer cells with PF-573228 resulted in FAK inactivation and changes in the expressions of lamin A/C and nuclear deformity. Since lamin A/C downregulation or deficiency was associated with cellular senescence, the senescence-associated ß-galactosidase (SA-ß-gal) assay was used to investigate whether PF-573228 treatment drove cellular senescence, which showed more SA-ß-gal-positive cells in culture. p53 is known to play a pivotal role in mediating the progression of cellular senescence, and the PF-573228-treated lung cancer cells resulted in a higher p53 expression level. Subsequently, the FAK depletion in lung cancer cells was employed to confirm the role of FAK inhibition on cellular senescence. FAK depletion and pharmacological inhibition of lung cancer cells elicited similar patterns of cellular senescence, lamin A/C downregulation, and p53 upregulation, implying that FAK signaling is associated with the expression of p53 and the maintenance of lamin A/C levels to shape regular nuclear morphology and manage anti-senescence. Conversely, FAK inactivation led to p53 upregulation, disorganization of the nuclear matrix, and consequently cellular senescence. Our data suggest a new FAK signaling pathway, in that abolishing FAK signaling can activate the senescence program in cells. Triggering cellular senescence could be a new therapeutic approach to limit tumor growth.

Extendin-4 protects kidney from acute ischemia-reperfusion injury through upregulation of NRF2 signaling.

This study tested the hypothesis that exendin-4 (Ex4) protects kidneys against ischemia-reperfusion (IR) injury mainly through upregulation of nuclear-factor erythroid 2-related factor 2 (Nrf2) signaling and downregulation of oxidative stress. Male-adult Sprague-Dawley rats (n=24) were equally divided into group 1 (sham-operated control), group 2 [IR only, ischemia (1 h)/reperfusion (72 h)] and group 3 (IR-Ex4, 10 µg/kg at 30 min, 24 h, 48 h after IR procedure). The in vitro study demonstrated that the protein expressions of phosphorylated (p)-Akt and Nrf2 were significantly progressively increased at time points of 0/0.5/1/3 h and 0/0.5/1/3/6/12/24 h, respectively in NRK-52E cells co-cultured with Ex4 (20 nM) (all P<0.0001). Additionally, the protein expressions of NOX-1/NOX2 were significantly increased, whereas p-Akt was significantly decreased in NRK-52E cells co-cultured with P-cresol (200 µM) that were significantly reversed after Ex4 treatment (all P<0.0001). As compared with baseline, the creatinine level, left/right kidney weight and MCP-1-positively stained area in the kidney parenchyma were significantly increased at 24 h after the IR procedure and significantly progressively decreased after that (all P<0.0001). By 27 h after IR, creatinine level/MCP-1 + area was significantly higher in group 2 than in groups 1 and 3, and significantly higher in group 3 than in group 1 (all P<0.0001). The numbers of Nrf2 +/NQO-1 + cells/SOD activity in kidney parenchyma were significantly lower in group 2 than in groups 1 and 3, and significantly lower in group 1 than in group 3 (all P<0.0001). In conclusion, Ex4 protected kidney from IR injury through upregulating antioxidants and downregulating inflammation/oxidative stress.

The mTOR-FAK mechanotransduction signaling axis for focal adhesion maturation and cell proliferation.

BACKGROUND: Mechanotransduction (MTD) is an important physiopathological signalling pathway associated with cardiovascular disease such as hypertension. Phosphorylation of focal adhesion kinase (FAK) is a MTD-sensing protein. This study tested the hypothesis that mTOR-FAK MTD signaling axis was crucial for focal adhesion (FA) maturation and cell proliferation. METHODS: Shock-wave was adopted as a tool for MTD and mTOR-FAK signaling. RESULTS: After demonstrating a failure in FAK phosphorylation after microfilament depolymerization, we attempted to identify the upstream regulator out of three kinases known to be activated in pressure-stimulated MTD [i.e., GSK-3ß, Akt, and mTORC1 (mammalian target of rapamycin complex 1)]. Of the three specific inhibitors, only rapamycin, an inhibitor of mTORC1, was found to inhibit FAK phosphorylation, suggesting that mTORC1 is the upstream regulator in shock-wave-elicited FAK phosphorylation. Moreover, mTOR and its readout protein S6K were found to be activated by shock-wave stimulation. On the other hand, microscopic examination revealed not only MTD-induced increase in the number of actin stress fibers, but also alternative subcellular localization of mTORC1 as vesicle-like inclusions on microfilaments. Besides, rapamycin was found to destruct the granular pattern of mTORC1, while dissociation between F-actin and mTORC1 was noted after cytochalasin D administration. Since mTORC1 and FAK are essential for cell proliferation, we performed proliferation assay for mesenchymal stem cell (MSC) with and without shock-wave administration/rapamycin treatment/FAK depletion. The results demonstrated significant enhancement of cell proliferation after shock-wave stimulation but remarkable suppression after rapamycin and siFAK treatment. CONCLUSION: Our findings suggest not only a co-ordinated regulation of FAK phosphorylation by mTORC1 and microfilaments, but also the participation of mTORC1-FAK signalling in MSC proliferation.

Severe bilateral ischemic-reperfusion renal injury: hyperacute and acute changes in apparent diffusion coefficient, T1, and T2 mapping with immunohistochemical correlations.

The aim of this study was to investigate the hyperacute and acute changes in apparent diffusion coefficient (ADC), T1, and T2 mapping in rat kidneys after severe bilateral renal ischemic-reperfusion injury (IRI). After baseline MRI, 24 Spraque-Dawley rats with renal IRI were divided equally as group 1 (post-IRI MRI at 6 hours, days 1, 3, and 7) and groups 2, 3, and 4 (post-IRI MRI at 6 hours; 6 hours and day 1; 6 hours, days 1 and 3, respectively), while six other rats without IRI (group 5) were used as sham control. ADC, T1, and T2 values of the cortex and outer and inner stripes of outer medulla (OSOM and ISOM), and immunohistochemical studies assessing monocyte chemoattractant protein-1 (MCP-1), CD68+ cells, tubular cast formation, and collagen deposition in three zones at different time points were evaluated. Significantly reduced ADCs in OSOM and ISOM are noninvasive biomarkers denoting hyperacute damages after IRI. Linear regression analysis revealed a significant inverse correlation between 6-hour/baseline ADC ratios and MCP-1 staining (P < 0.001, r2 = 0.738). ADC, T1, and T2 values are useful for assessing variable IRI changes in different layers depending on underlying microstructural and histopathological changes at different time points.

LIMCH1 regulates nonmuscle myosin-II activity and suppresses cell migration.

Nonmuscle myosin II (NM-II) is an important motor protein involved in cell migration. Incorporation of NM-II into actin stress fiber provides a traction force to promote actin retrograde flow and focal adhesion assembly. However, the components involved in regulation of NM-II activity are not well understood. Here we identified a novel actin stress fiber-associated protein, LIM and calponin-homology domains 1 (LIMCH1), which regulates NM-II activity. The recruitment of LIMCH1 into contractile stress fibers revealed its localization complementary to actinin-1. LIMCH1 interacted with NM-IIA, but not NM-IIB, independent of the inhibition of myosin ATPase activity with blebbistatin. Moreover, the N-terminus of LIMCH1 binds to the head region of NM-IIA. Depletion of LIMCH1 attenuated myosin regulatory light chain (MRLC) diphosphorylation in HeLa cells, which was restored by reexpression of small interfering RNA-resistant LIMCH1. In addition, LIMCH1-depleted HeLa cells exhibited a decrease in the number of actin stress fibers and focal adhesions, leading to enhanced cell migration. Collectively, our data suggest that LIMCH1 plays a positive role in regulation of NM-II activity through effects on MRLC during cell migration.

Assessment of doxorubicin-induced mouse testicular damage by the novel second-harmonic generation microscopy.

Microtubules, maintaining a non-linear structure, are suitable for direct observation in living mammalian by second-harmonic imaging microscopy (SHIM) (a new kind of confocal microscopies). Testes constituted by vast seminiferous microtubules (SM), serve as good candidates for visualization by SHIM. This study employs the SHIM and Western-blot (WB) to assess the cellular-molecular levels of doxorubicin (Dox)-induced mouse testicular damage. The SHIM examination was able to clearly identify the integrity of normal architecture of the living mouse testis, namely, the anatomical features of SM, smooth muscle wall of SM, manchette microtubules, exoplasmic microtubules in Sertoli cells and interstitial connective tissue, as well as the destructive feature of SM in Dox-treated mice (n = 6 per group). By day 21 after Dox-treatment, the testicular weight and testicular length were significantly progressively decreased as Dox dosage was stepwise increased, i.e., 0/5/10/15/20 mg/kg/body-weight (BW) (all p<0.0001). The cross-section area of SM was significantly lower in Dox-treated (15 mg/kg-BW) mice than that in controls (p<0.001). The protein expression of vimentin was significantly progressively increased whereas the protein expression of ß-tubulin/androgen-receptor was significantly progressively decreased in stepwise increased Dox dosage (all p<0.001). The protein expressions of inflammatory (MMP-9/IL-1ß/TNF-α/iNOX), oxidative-stress (NOX-1/NOX-2/NOX-4/oxidized protein), apoptotic (mitochondrial-Bax/cleaved-caspase-3/PARP), fibrotic (Smad3/TGF-ß) mitochondrial/DNA-damaged (cytosolic cytochrome-C/γ-H2AX/ATM/KU70), and cell apoptotic/death (PTEN/p53) biomarkers were significantly higher in Dox-treated (15 mg/kg-BW) group than those in controls (all p<0.001). In conlusion, the dose-dependent Dox-caused mouse testicular damage can be not only detected by WB in molecular level but also clearly identified by SHIM in living mice.

Dynamin-Related Protein 1 Promotes Mitochondrial Fission and Contributes to The Hippocampal Neuronal Cell Death Following Experimental Status Epilepticus.

AIMS: Prolonged seizure activity may result in mitochondrial dysfunction and lead to cell death in the hippocampus. Mitochondrial fission may occur in an early stage of neuronal cell death. This study examined the role of the mitochondrial fission protein dynamin-related protein 1 (Drp1) in the hippocampus following status epilepticus. METHODS: Kainic acid (KA) was microinjected unilaterally into the hippocampal CA3 area in Sprague Dawley rats to induce prolonged seizure activity. Biochemical analysis, electron microscopy, and immunofluorescence staining were performed to evaluate the subsequent molecular and cellular events. The effects of pretreatment with a mitochondrial fission protein inhibitor, Mdivi-1 (2 nmol), were also evaluated. RESULTS: Phosphorylation of Drp1 at serine 616 (p-Drp1(Ser616)) was elevated from 1 to 24 h after the elicited seizure activity. Pretreatment with Mdivi-1 decreased the Drp1 phosphorylation at Ser616 and limited the mitochondrial fission. Mdivi-1 rescued the Complex I dysfunction, decreased the levels of oxidized proteins, decreased the activation of cytochrome c/caspase-3 signaling, and blunted cell death in CA3 neurons. CONCLUSION: Our findings suggest that activation of p-Drp1(Ser616) is related to seizure-induced neuronal damage. Modulation of p-Drp1(Ser616) expression is accompanied by decreases in mitochondrial fission, mitochondrial dysfunction, and oxidation, providing a neuroprotective effect against seizure-induced hippocampal neuronal damage.

Exendin-4 therapy still offered an additional benefit on reducing transverse aortic constriction-induced cardiac hypertrophy-caused myocardial damage in DPP-4 deficient rats.

Inhibition of dipeptidyl peptidase-IV (DPP-4) enzyme activity has been revealed to protect myocardium from ischemia-reperfusion through enhancing the endogenous glucagon-like peptide-1 (GLP-1) level. However, whether exogenous supply of exendin-4, an analogue of GLP-1, would still offer benefit for protecting myocardial damage from trans-aortic constriction (TAC)-induced hypertrophic cardiomyopathy in preexistence of DPP-4 deficiency (DPP-4(D)) remained unclear. Male-adult (DPP-4(D)) rats (n = 32) were randomized into group 1 [sham control (SC)], group 2 (DPP-4(D) + TAC), group 3 [DPP-4(D) + TAC + exendin-4 10 µg/day], and group 4 [DPP-4(D) + TAC + exendin-4 10 µg + exendin-9-39 10 µg/day]. The rats were sacrificed by day 60 after last echocardiographic examination. By day 60 after TAC, left ventricular ejection fraction (LVEF) (%) was highest in group 1 and lowest in group 2, and significantly lower in group 4 than that in group 3 (all p < 0.001). The protein expressions of oxidative stress (oxidized protein, NOX-1, NOX-2), inflammatory (MMP-9, TNF-α, NF-κB), apoptotic (Bax, cleaved caspase 3 and PARP), fibrotic (TGF-ß, Smad3), heart failure (BNP, ß-MHC), DNA damaged (γ-H2AX) and ischemic stress (p-P38, p-Akt, p53, ATM) biomarkers showed an opposite pattern of LVEF among the four groups (all p < 0.03). Fibrotic area (by Masson's trichrome, Sirius red), and cellular expressions of DNA-damaged markers (Ki-67+, γ-H2AX+, CD90+/53BP1+) displayed an identical pattern, whereas cellular expressions of angiogenesis (CD31+, α-SMA+) and sarcomere length exhibited an opposite pattern compared to that of oxidative stress among the four groups (all p < 0.001). Take altogether, Exendin-4 effectively suppressed TAC-induced pathological cardiac hypertrophy in DPP-4(D) rat.