Suppression of hypoxia-induced CAV1 autophagic degradation enhances nanoalbumin-paclitaxel transcytosis and improves therapeutic activity in pancreatic cancer.

Nanoalbumin-paclitaxel (nab-paclitaxel) is a standard chemotherapy for pancreatic cancer but has shown limited efficacy. However, the mechanism through which circulating nab-paclitaxel passes through the tumour vascular endothelium has not been determined. In our study, a new nonradioactive and highly sensitive method for analysing nab-paclitaxel transcytosis was established. Based on these methods, we found that hypoxia significantly enhanced the autophagic degradation of CAV1 and therefore attenuated caveolae-mediated nab-paclitaxel transcytosis across endothelial cells (ECs). In a proof-of-concept experiment, higher levels of CAV1, accompanied by lower levels of LC3B, were observed in the vascular endothelium of pancreatic cancer tissues collected from patients who showed a good response to nab-paclitaxel compared with those from patients who showed a poor response to nab-paclitaxel. Furthermore, both in vivo and in vitro studies confirmed that suppressing the autophagic degradation of CAV1 via EC-specific ATG5 knockdown or hydroxychloroquine sulfate (HCQ) treatment significantly enhanced nab-paclitaxel translocation across the endothelial barrier into pancreatic cancer cells and amplified the inhibitory effect of nab-paclitaxel on pancreatic tumour growth. The stimulation of CAV1 expression by EC-specific overexpression of exogenous CAV1 or administration of gemcitabine hydrochloride (GE) had the same effect. These results demonstrated that suppressing CAV1 autophagic degradation is a novel translatable strategy for enhancing nab-paclitaxel chemotherapeutic activity in the treatment of pancreatic cancer.

AGEs promote atherosclerosis by increasing LDL transcytosis across endothelial cells via RAGE/NF-κB/Caveolin-1 pathway.

OBJECTIVE: To elucidate the mechanism whereby advanced glycation end products (AGEs) accelerate atherosclerosis (AS) and to explore novel therapeutic strategies for atherosclerotic cardiovascular disease. METHODS AND RESULTS: The effect of AGEs on low-density lipoprotein (LDL) transcytosis across endothelial cells (ECs) was assessed using an in vitro model of LDL transcytosis. We observed that AGEs activated the receptor for advanced glycation end products (RAGE) on the surface of ECs and consequently upregulated Caveolin-1, which in turn increased caveolae-mediated LDL transcytosis and accelerated AS progression. Our molecular assessment revealed that AGEs activate the RAGE-NF-κB signaling, which then recruits the NF-κB subunit p65 to the RAGE promoter and consequently enhances RAGE transcription, thereby forming a positive feedback loop between the NF-κB signaling and RAGE expression. Increased NF-κB signaling ultimately upregulated Caveolin-1, promoting LDL transcytosis, and inhibition of RAGE suppressed AGE-induced LDL transcytosis. In ApoE-/- mice on a high-fat diet, atherosclerotic plaque formation was accelerated by AGEs but suppressed by EC-specific knockdown of RAGE. CONCLUSION: AGEs accelerate the development of diabetes-related AS by increasing the LDL transcytosis in ECs through the activation of the RAGE/NF-κB/Caveolin-1 axis, which may be targeted to prevent or treat diabetic macrovascular complications.

Serum amyloid A and interleukin -1ß facilitate LDL transcytosis across endothelial cells and atherosclerosis via NF-κB/caveolin-1/cavin-1 pathway.

BACKGROUND AND AIMS: Inflammatory molecules play important roles in atherosclerosis. We aimed to illustrate the roles of serum amyloid A (SAA), and interleukin (IL)-1ß in low density lipoproteins (LDL) transcytosis and atherosclerosis. METHODS: Effects of SAA and IL-1ß on transcytosis of LDL were measured by an in vitro LDL transcytosis model. NF-κB/caveolin-1/cavin-1 pathway activation was investigated by Western blots and ELISA. Effects of SAA and IL-1ß on the retention of LDL in aorta of C57BL/6J mice were detected by IVIS spectrum. Effects of SAA and IL-1ß on atherosclerosis in Apoe-/- mice were examined by Oil Red O staining. RESULTS: SAA and IL-1ß stimulated LDL transcytosis across endothelial cells (ECs), which was accompanied by an increase in LDL uptake by ECs. SAA and IL-1ß enhanced the activity of nuclear factor (NF)-κB, consequently facilitating an up-regulation of proteins involved in caveolae formation, including caveolin-1 and cavin-1, along with an assembly of NLRP3 inflammasome. Furthermore, SAA- and IL-1ß-induced effects were blocked by NF-κB subunit p65 siRNA. Meanwhile, SAA- and IL-1ß-induced LDL transcytosis were effectively blocked by caveolin-1 siRNA or cavin-1 siRNA. Interestingly, SAA and IL-1ß facilitated LDL entering into the aorta of C57BL/6J mice. In Apoe-/- mice, SAA and IL-1ß increased the areas of lipid-rich atherosclerotic lesions in the both ascending and root of aorta. Furthermore, a significant increase in the NLRP3 inflammasome, accompanied by accumulation of cavin-1 and caveolin-1, was observed in the aortic endothelium of Apoe-/- mice. CONCLUSIONS: SAA and IL-1ß accelerated LDL transcytosis via the NF-κB/caveolin-1/cavin-1 axis.

Remnant cholesterol is independently asssociated with an increased risk of peripheral artery disease in type 2 diabetic patients.

Background: Remnant cholesterol (RC) has been correlated with a higher risk of atherosclerosis. It has been confirmed that in the general population, an elevated RC level is related to a 5-fold higher risk of peripheral arterial disease (PAD). Diabetes is one of the strongest risk factors for PAD development. However, the association between RC and PAD in the specific population of type 2 diabetes mellitus (T2DM) has not been investigated. Herein, the correlation was investigated between RC and PAD in T2DM patients. Methods: In the retrospective study, the hematological parameter data of 246 T2DM patients without PAD (T2DM - WPAD) and 270 T2DM patients with PAD (T2DM - PAD) was collected. Differences in RC levels between the two groups were compared, and the association between RC and PAD severity was examined. Multifactorial regression was used to determine whether RC was a significant contributor to the development of T2DM - PAD. The diagnostic potential of RC was tested using receiver operating characteristic (ROC) curve. Results: The RC levels in T2DM - PAD individuals were considerably greater than in T2DM - WPAD individuals (P < 0.001). RC had a positive correlation with disease severity. Further, multifactorial logistic regression analyses found that elevated RC levels were a major contributor to T2DM - PAD (P < 0.001). The area under the curve (AUC) of the RC for T2DM - PAD patients was 0.727. The cut-off value of RC was 0.64 mmol/L. Conclusion: The RC levels were higher in T2DM - PAD patients, and were independently linked with its severity. Diabetic patients with RC levels > 0.64 mmol/L had an elevated risk of developing PAD.

Scavenger receptor a mediates glycated LDL transcytosis across endothelial cells to promote atherosclerosis.

Glycated low-density lipoprotein (G-LDL) is an established proatherosclerotic factor, but the mechanism is not completely understood. In vitro, we evaluated the uptake and transcytosis rates of N-LDL and G-LDL in endothelial cells and the uptake and transcytosis rates of G-LDL were much higher than those of N-LDL. Then, using small interfering RNAs, the receptor mediating G-LDL uptake and transcytosis was screened among eight candidate receptors, and the mechanism of the receptor regulation was thoroughly examined. We discovered that scavenger receptor A (SR-A) knockdown dramatically decreased the uptake and transcytosis rates of G-LDL. Additionally, endothelial cells with overexpressed SR-A had enhanced G-LDL uptake and transcytosis. In vivo, G-LDL was injected in the tail vein of ApoE-/- mice to investigate whether G-LDL affects atherosclerotic plaque formation. Compared with the injection of N-LDL, the injection of G-LDL accelerated atherosclerotic plaque formation in ApoE-/- mice, which was ameliorated by endothelial cells specific SR-A knockdown. Together, our results provide the first demonstration that the transcytosis of G-LDL across endothelial cells is much faster than that of N-LDL and SR-A is the major type of receptor responsible for G-LDL binding and transcytosis across endothelial cells.

VCAM-1-binding peptide targeted cationic liposomes containing NLRP3 siRNA to modulate LDL transcytosis as a novel therapy for experimental atherosclerosis.

BACKGROUND: Activation of NLRP3 inflammasome accelerates the formation of atherosclerotic plaques. Here, we evaluated the effects of inflammation on the expression of the NLRP3 inflammasome in endothelial cells (ECs). METHODS: The effect of TNF-α on transcytosis of LDL was measured. VCAM-1 binding peptide targeting cationic liposomes (PCLs) were prepared as siRNA vectors. Methylated NLRP3 siRNA was encapsulated into the PCLs to knock down NLRP3 in vitro and in vivo. In rats with partial carotid ligation, TNF-α-induced LDL retention in the carotid artery endothelium was observed. In ApoE-/- mice, NLRP3 siRNA-PCLs were injected intravenously to observe their effect on the formation of atherosclerosis. RESULTS: Our results showed that TNF-α upregulated NLRP3 in ECs, promoting the assembly of the NLRP3 inflammasome and processing of pro-IL-1ß into IL-1ß. Moreover, TNF-α accelerated LDL transcytosis in ECs. Knockdown of NLRP3 prevented TNF-α-induced NLPR3 inflammasome/IL-1ß signaling and LDL transcytosis. Using optimized cationic liposomes to encapsulate methylated NLRP3 siRNA, resulting in targeting of VCAM-1-expressing ECs, to knockdown NLRP3, TNF-α-induced NLRP3 inflammasome activation and LDL transcytosis were prevented. Using the partial carotid ligation as an atherosclerosis rat model, we found that local administration of NLRP3 siRNA-PCLs efficiently knocked down NLPR3 expression in the carotid endothelium and dramatically attenuated the deposition of atherogenic LDL in carotid ECs in TNF-α-challenged rats. Furthermore, NLRP3 siRNA-PCLs were injected intravenously in ApoE-/- mice, resulting in reduced plaque formation. CONCLUSION: These findings established a novel strategy for targeting the NLRP3 inflammasome using NLRP3 siRNA-PCLs to interrupt LDL transcytosis, representing a potential novel therapy for atherosclerosis.

Deacetylation of Caveolin-1 by Sirt6 induces autophagy and retards high glucose-stimulated LDL transcytosis and atherosclerosis formation.

BACKGROUND: Atherosclerosis (AS) is the basis of diabetic macrovascular complications. The plasma low-density lipoprotein (LDL) particles transcytosis across endothelial cells (ECs) and deposition under the endothelium is the initiation step of AS. We previously reported that high glucose inhibits the autophagic degradation of Caveolin-1 and promote LDL transcytosis across ECs, which in turn accelerates atherosclerotic progression. Since Sirt6 is a chromatin-associated protein with deacetylation activity, whether it can regulate Caveolin-1 acetylation and regulating the autophagic degradation of Caveolin-1 remains elusive. METHODS: Autophagy and histone acetylation were assessed in the umbilical cords of patients with gestational diabetes mellitus (GDM) by immunohistochemistry. An in vitro model of LDL transcytosis was established, and the role of Sirt6 in LDL transcytosis across endothelial cells was clarified. The effect of Sirt6 on the autophagic degradation of Caveolin-1 under hyperglycemic conditions was explored in a streptozotocin (STZ)-induced diabetic AS model established using the ApoE-/- mice. RESULTS: Caveolin-1 and acetylated histone H3 levels were significantly increased, while LC3B and Sirt6 were downregulated in the monolayer of the vascular wall from GDM and type 2 diabetes mellitus (T2DM) patients. Immunoprecipitation assays showed that Sirt6 interacts with Caveolin-1 and specifically mediated its acetylation levels. Immuno-electron microscopy (EM) further indicated that Sirt6 overexpression triggered the autophagic lysosomal degradation of Caveolin-1. ECs-specific overexpression of Sirt6 by adeno-associated viral vector serotype 9 (AAV9) induced autophagy, reduced Caveolin-1 expression, and ameliorated atherosclerotic plaque formation in STZ-induced diabetic ApoE-/- mice. CONCLUSION: Sirt6-mediated acetylation of Caveolin-1 activates its autophagic degradation and inhibits high glucose-stimulated LDL transcytosis. Thus, the Sirt6/Caveolin-1 autophagic pathway plays a crucial role in diabetic AS, and the overexpression or activation of Sirt6 is a novel therapeutic strategy.

Critical role of minor eggcase silk component in promoting spidroin chain alignment and strong fiber formation.

Natural spider silk with extraordinary mechanical properties is typically spun from more than one type of spidroin. Although the main components of various spider silks have been widely studied, little is known about the molecular role of the minor silk components in spidroin self-assembly and fiber formation. Here, we show that the minor component of spider eggcase silk, TuSp2, not only accelerates self-assembly but remarkably promotes molecular chain alignment of spidroins upon physical shearing. NMR structure of the repetitive domain of TuSp2 reveals that its dimeric structure with unique charged surface serves as a platform to recruit different domains of the main eggcase component TuSp1. Artificial fiber spun from the complex between TuSp1 and TuSp2 minispidroins exhibits considerably higher strength and Young's modulus than its native counterpart. These results create a framework for rationally designing silk biomaterials based on distinct roles of silk components.

Age-related changes of human serum Sirtuin6 in adults.

BACKGROUND: Aging is a natural life process and with an aging population, age-related diseases (e.g. type 2 diabetes mellitus (T2DM), atherosclerosis-based cardiovascular diseases) are the primary mortality cause in older adults. Telomerase is often used as an aging biomarker. Detection and characterization of novel biomarkers can help in a more specific and sensitive identification of a person's aging status. Also, this could help in age-related diseases early prevent, ultimately prolonging the population's life span. Sirtuin 6 (Sirt6) - a member of the Sirtuins NAD+-dependent histone deacetylases family - is mainly intracellularly expressed, and is reported to be involved in the regulation of aging and aging-related diseases. Whether serum Sirt6 is correlated with aging and could be used as an aging biomarker is unknown. In the present study, we aimed to investigate the age-related Sirt6 changes in the serum of human adults. METHODS: Participants were divided into three groups according to age: 20-30 years (Young); 45-55 years (Middle-aged); and ≥ 70 years (Old). The Sirt6 and telomerase serum concentrations were determined by ELISA. The Sirt6 and human telomerase reverse transcriptase (hTERT) expression in vessels from amputated human lower limbs were analyzed using real-time quantitative PCR (RT-qPCR) and immunohistochemical staining. The relationships between variables were evaluated by Pearson correlation analysis. RESULTS: The Sirt6 and telomerase serum levels reduced with an increase in age. A similar tendency was observed for Sirt6 and hTERT in the vessel. Serum levels of Sirt6 were higher in females compared with males. Pearson's regression analysis revealed that the Sirt6 serum level positively correlated with telomerase (r = 0.5743) and both were significantly negatively correlated with age (r = - 0.5830 and r = - 0.5993, respectively). CONCLUSIONS: We reported a negative correlation between serum Sirt6 concentration and aging in human beings. Therefore, the Sirt6 serum level is a potential sex-specific aging marker.

Leptin gene-targeted editing in ob/ob mouse adipose tissue based on the CRISPR/Cas9 system.

Gene therapy has become the most effective treatment for monogenic diseases. Congenital LEPTIN deficiency is a rare autosomal recessive monogenic obesity syndrome caused by mutations in the Leptin gene. Ob/ob mouse is a monogenic obesity model, which carries a homozygous point mutation of C to T in Exon 2 of the Leptin gene. Here, we attempted to edit the mutated Leptin gene in ob/ob mice preadipocytes and inguinal adipose tissues using CRISPR/Cas9 to correct the C to T mutation and restore the production of LEPTIN protein by adipocytes. The edited preadipocytes exhibit a correction of 5.5% of Leptin alleles and produce normal LEPTIN protein when differentiated into mature adipocytes. The ob/ob mice display correction of 1.67% of Leptin alleles, which is sufficient to restore the production and physiological functions of LEPTIN protein, such as suppressing appetite and alleviating insulin resistance. Our study suggests CRISPR/Cas9-mediated in situ genome editing as a feasible therapeutic strategy for human monogenic diseases, and paves the way for further research on efficient delivery system in potential future clinical application.

[Formation of natural silk and progress in artificial spinning].

Natural silks, produced by spiders and silkworms, are excellent materials with marvelous mechanical properties, biocompatibility and biodegradability, and widely used in the fields of textile, optics, electronics, biomedicine and environmental engineering. So far, there are many spinning methods to improve the mechanical properties of artificial fibers, such as wet spinning, dry spinning, and electrospinning. However, the performance of most artificial fibers is still inferior to natural silks. It is important to understand the correlations between hierarchical structures and performance in the field of artificial spinning. This review introduces the formation of natural silks, the relationship between the mechanical properties of silks and the hierarchical structure, the research progress in artificial spinning, and the application of silks.

Multidimensional Analysis of Risk Factors for the Severity and Mortality of Patients with COVID-19 and Diabetes.

INTRODUCTION: Diabetes is one of the most common comorbidities of COVID-19. We aimed to conduct a multidimensional analysis of risk factors associated with the severity and mortality of patients with COVID-19 and diabetes. METHODS: In this retrospective study involving 1443 patients with COVID-19, we analyzed the clinical and laboratory characteristics and risk factors associated with disease severity in patients with COVID-19 with and without diabetes. Binary logistic regression analyses were performed to identify the risk factors associated with mortality in patients with COVID-19 and diabetes. The 84-day survival duration for critical patients with COVID-19 and diabetes who had different levels of leukocytes and neutrophils, or treated with immunoglobulin or not, was conducted using Kaplan-Meier survival curves. RESULTS: Of the 1443 patients with COVID-19, 256 (17.7%) had diabetes, had a median age of 66.0 [IQR 58.0-73.8] years, and were more likely to develop severe (41.8% vs. 35.6%) and critical disease (34.0% vs. 14.9%), followed by higher mortality (21.1% vs. 7.0%), than those without diabetes. Higher levels of leukocytes (> 5.37 × 109/L), older age, and comorbid cerebrovascular disease and chronic renal disease independently contributed to in-hospital death of patients with COVID-19 and diabetes. Leukocytes > 5.37 × 109/L and the application of immunoglobulin were associated with shorter survival duration and lower mortality, respectively, in critical patients with COVID-19 and diabetes. CONCLUSIONS: More attention should be paid to patients with COVID-19 and diabetes, especially when they have high leukocyte counts (> 5.37 × 109/L). Timely and adequate intravenous immunoglobulin (IVIG) use may reduce the mortality of critical patients with COVID-19 and diabetes.

Salidroside-Mediated Autophagic Targeting of Active Src and Caveolin-1 Suppresses Low-Density Lipoprotein Transcytosis across Endothelial Cells.

Subendothelial retention of apolipoprotein B100-containing lipoprotein, such as low-density lipoprotein (LDL), is the initial step of atherogenesis. Activation of autophagy exhibits beneficial effects for the treatment of atherosclerosis. In our previous study, we demonstrated that hyperglycemia suppressed autophagic degradation of caveolin-1, which in turn resulted in acceleration of caveolae-mediated LDL transcytosis across endothelial cells and lipid retention. Therefore, targeting the crossed pathway in autophagy activation and LDL transcytosis interruption may be a promising antiatherosclerotic strategy. In metabolic diseases, including atherosclerosis, salidroside, a phenylpropanoid glycoside compound (3,5-dimethoxyphenyl) methyl-ß-glucopyranoside), is the most important compound responsible for the therapeutic activities of Rhodiola. However, whether salidroside suppresses LDL transcytosis to alleviate atherosclerosis has not yet been elucidated. In the present study, we demonstrated that salidroside significantly decreased LDL transcytosis across endothelial cells. Salidroside-induced effects were dramatically blocked by AMPK (adenosine monophosphate-activated protein kinase) inhibitor (compound c, AMPKα siRNA) and by overexpression of exogenous tyrosine-phosphorylated caveolin-1 using transfected cells with phosphomimicking caveolin-1 on tyrosine 14 mutant plasmids (Y14D). Furthermore, we observed that salidroside promoted autophagosome formation via activating AMPK. Meanwhile, the interaction between caveolin-1 and LC3B-II, as well as the interaction between active Src (indicated by the phosphorylation of Src on tyrosine 416) and LC3B-II, was significantly increased, upon stimulation with salidroside. In addition, both bafilomycin A1 (a lysosome inhibitor) and an AMPK inhibitor (compound c) markedly prevented salidroside-induced autophagic degradation of p-Src and caveolin-1. Moreover, the phosphorylation of caveolin-1 on tyrosine 14 was disrupted due to the downregulation of p-Src and caveolin-1, thereby directly decreasing LDL transcytosis by attenuating the number of caveolae on the cell membrane and by preventing caveolae-mediated LDL endocytosis released from the cell membrane. In ApoE-/- mice, salidroside significantly delayed the formation of atherosclerotic lesions. Meanwhile, a significant increase in LC3B, accompanied by attenuated accumulation of the autophagy substrate SQSTM1, was observed in aortic endothelium of ApoE-/- mice. Taken together, our findings demonstrated that salidroside protected against atherosclerosis by inhibiting LDL transcytosis through enhancing the autophagic degradation of active Src and caveolin-1.

CAV1-CAVIN1-LC3B-mediated autophagy regulates high glucose-stimulated LDL transcytosis.

Diabetes is a recognized high-risk factor for the development of atherosclerosis, in which macroautophagy/autophagy is emerging to play essential roles. The retention of low-density lipoprotein (LDL) particles in subendothelial space following transcytosis across the endothelium is the initial step of atherosclerosis. Here, we identified that high glucose could promote atherosclerosis by stimulating transcytosis of LDL. By inhibiting AMPK-MTOR-PIK3C3 pathway, high glucose suppresses the CAV-CAVIN-LC3B-mediated autophagic degradation of CAV1; therefore, more CAV1 is accumulated in the cytosol and utilized to form more caveolae in the cell membrane and facilitates the LDL transcytosis across endothelial cells. For a proof of concept, higher levels of lipids were accumulated in the subendothelial space of umbilical venous walls from pregnant women with gestational diabetes mellitus (GDM), compared to those of pregnant women without GDM. Our results reveal that high glucose stimulates LDL transcytosis by a novel CAV1-CAVIN1-LC3B signaling-mediated autophagic degradation pathway. ABBREVIATIONS: 3-MA: 3-methyladenine; ACTB: actin beta; AMPK: AMP-activated protein kinase; Bafi: bafilomycin A1; CAV1: caveolin-1; CAVIN1: caveolae associated protein 1; CSD: the CAV1 scaffolding domain; GDM: gestational diabetes mellitus; IMD: intramembrane domain; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule- associated protein 1 light chain 3; MFI: mean fluorescence intensity; MTOR: mechanistic target of rapamycin kinase; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; SQSTM1/p62: sequestosome 1.

Rhodiola and salidroside in the treatment of metabolic disorders.

Over the past three decades, the knowledge gained about the mechanisms that underpin the potential use of Rhodiola in stress- and ageing-associated disorders has increased, and provided a universal framework for studies that focused on the use of Rhodiola in preventing or curing metabolic diseases. Of particular interest is the emerging role of Rhodiola in the maintenance of energy homeostasis. Moreover, over the last two decades, great efforts have been undertaken to unravel the underlying mechanisms of action of Rhodiola in the treatment of metabolic disorders. Extracts of Rhodiola and salidroside, the most abundant active compound in Rhodiola, are suggested to provide a beneficial effect in mental, behavioral, and metabolic disorders. Both in vivo and ex vivo studies, Rhodiola extracts and salidroside ameliorate metabolic disorders when administered acutely or prior to experimental injury. The mechanism involved includes multi-target effects by modulating various synergistic pathways that control oxidative stress, inflammation, mitochondria, autophagy, and cell death, as well as AMPK signaling that is associated with possible beneficial effects on metabolic disorders. However, evidence-based data supporting the effectiveness of Rhodiola or salidroside in treating metabolic disorders is limited. Therefore, a comprehensive review of available trials showing putative treatment strategies of metabolic disorders that include both clinical effective perspectives and fundamental molecular mechanisms is warranted. This review highlights studies that focus on the potential role of Rhodiola extracts and salidroside in type 2 diabetes and atherosclerosis, the two most common metabolic diseases.

Low density lipoprotein mimics insulin action on autophagy and glucose uptake in endothelial cells.

Elevated plasma low density lipoprotein (LDL) is an established risk factor for cardiovascular disease. In addition to being able to cross the endothelial barrier to become accumulated in subendothelial space and thereby initiate atherosclerosis, LDL may exert a direct effect on vascular endothelial cells through activation of LDL receptor and its downstream signaling. Whether LDL can modulate the signaling for autophagy in endothelial cells is not clear. The present study firstly demonstrated that LDL can suppress endothelial autophagy through activation of the PI3K/Akt/mTOR signaling pathway and can promote glucose uptake by translocating glucose transporter 1 (GLUT1) from cytoplasm to cell membrane, actions similar to those of insulin. A co-immunoprecipitation assay found that LDL receptor (LDLR) and insulin receptor (IR) formed a complex in HUVECs. Knock down of the insulin receptor by small interfering RNA blocked the suppression of autophagy by LDL, as well as the signaling pathway involved. We conclude that LDL may mimic the action of insulin in endothelial cells, which might partly explain the increased incidence of diabetes in patients receiving some LDL-lowering therapy.

Cavin-1 regulates caveolae-mediated LDL transcytosis: crosstalk in an AMPK/eNOS/ NF-κB/Sp1 loop.

Caveolae are specialized lipid rafts structure in the cell membrane and critical for regulating endothelial functions, e.g. transcytosis of macromolecules like low density lipoprotein (LDL) etc. Specifically, the organization and functions of caveolae are mediated by structure protein (caveolin-1) and adapter protein (cavin-1). The pathogenic role of caveolin-1 is well studied; nevertheless, mechanisms whereby cavin-1 regulates signaling transduction remain poorly understood. The aim of this study was designed to explore the role of cavin-1 in caveolae-mediated LDL transcytosis across endothelial cells. We reported here that cavin-1 knockdown mediated by small interfering RNA (siRNA) caused a significant decrease of LDL transcytosis. Moreover, cavin-1 knockdown increased the activity of endothelial nitric oxide synthase (eNOS) and the production of nitric oxide (NO). Consequently, an eNOS inhibitor, N-Nitro-L-Arginine Methyl Ester (L-NAME), not only suppressed the activity of specificity protein (Sp1) and nuclear factor kappa B (NF-κB), but also inhibited both activities via activating adenosine 5'-monophosphate- activated protein kinase (AMPK). In conclusion, we proposed an AMPK/eNOS/NF-κB/Sp1 circuit loop was formed to regulate caveolae residing proteins' expression, e.g. LDL receptor (LDLR), caveolin-1, eNOS, thereby to regulate caveolae-mediated LDL transcytosis in endothelial cells.

Siglec-5 is a novel marker of critical limb ischemia in patients with diabetes.

Critical Limb Ischemia (CLI) is common but uncommonly diagnosed. Improved recognition and early diagnostic markers for CLI are needed. Therefore, the aim of our study was to identify plasma biomarkers of CLI in patients with type 2 diabetes mellitus (T2DM). In this study, antibody-coated glass slide arrays were used to determine the plasma levels of 274 human cytokines in four matched cases of diabetes with and without CLI. Potential biomarkers were confirmed in an independent cohort by ELISA. After adjusting for confounding risk factors, only plasma level of Siglec-5 remained significantly associated with an increased odds ratio (OR) for diabetes with CLI by binary logistic regression analysis. Receiver operating characteristic (ROC) curve analysis revealed the optimal cut-off points for Siglec-5 was 153.1 ng/ml. After entering Siglec-5, the AUC was 0.99, which was higher than that of confounding risk factors only (AUC = 0.97, P < 0.05). Siglec-5 was expressed in plaques, but not in healthy artery wall in T2DM patients. Elevated plasma Siglec-5 was independently associated with CLI in T2DM. Plasma Siglec-5 levels are implicated as an early diagnostic marker of CLI in T2DM patients and it may become a target for the prevention or treatment of CLI in diabetes.

TNF-α stimulates endothelial palmitic acid transcytosis and promotes insulin resistance.

Persistent elevation of plasma TNF-α is a marker of low grade systemic inflammation. Palmitic acid (PA) is the most abundant type of saturated fatty acid in human body. PA is bound with albumin in plasma and could not pass through endothelial barrier freely. Albumin-bound PA has to be transported across monolayer endothelial cells through intracellular transcytosis, but not intercellular diffusion. In the present study, we discovered that TNF-α might stimulate PA transcytosis across cardiac microvascular endothelial cells, which further impaired the insulin-stimulated glucose uptake by cardiomyocytes and promoted insulin resistance. In this process, TNF-α-stimulated endothelial autophagy and NF-κB signaling crosstalk with each other and orchestrate the whole event, ultimately result in increased expression of fatty acid transporter protein 4 (FATP4) in endothelial cells and mediate the increased PA transcytosis across microvascular endothelial cells. Hopefully the present study discovered a novel missing link between low grade systemic inflammation and insulin resistance.

A novel mechanism of action for salidroside to alleviate diabetic albuminuria: effects on albumin transcytosis across glomerular endothelial cells.

Salidroside (SAL) is a phenylethanoid glycoside isolated from the medicinal plant Rhodiola rosea. R. rosea has been reported to have beneficial effects on diabetic nephropathy (DN) and high-glucose (HG)-induced mesangial cell proliferation. Given the importance of caveolin-1 (Cav-1) in transcytosis of albumin across the endothelial barrier, the present study was designed to elucidate whether SAL could inhibit Cav-1 phosphorylation and reduce the albumin transcytosis across glomerular endothelial cells (GECs) to alleviate diabetic albuminuria as well as to explore its upstream signaling pathway. To assess the therapeutic potential of SAL and the mechanisms involved in DN albuminuria, we orally administered SAL to db/db mice, and the effect of SAL on the albuminuria was measured. The albumin transcytosis across GECs was explored in a newly established in vitro cellular model. The ratio of albumin to creatinine was significantly reduced upon SAL treatment in db/db mice. SAL decreased the albumin transcytosis across GECs in both normoglycemic and hyperglycemic conditions. SAL reversed the HG-induced downregulation of AMP-activated protein kinase and upregulation of Src kinase and blocked the upregulation Cav-1 phosphorylation. Meanwhile, SAL decreased mitochondrial superoxide anion production and moderately depolarized mitochondrial membrane potential. We conclude that SAL exerts its proteinuria-alleviating effects by downregulation of Cav-1 phosphorylation and inhibition of albumin transcytosis across GECs. These studies provide the first evidence of interference with albumin transcytosis across GECs as a novel approach to the treatment of diabetic albuminuria.