CD151 Maintains Endolysosomal Protein Quality to Inhibit Vascular Inflammation.

BACKGROUND: Tetraspanin CD151 is highly expressed in endothelia and reinforces cell adhesion, but its role in vascular inflammation remains largely unknown. METHODS: In vitro molecular and cellular biological analyses on genetically modified endothelial cells, in vivo vascular biological analyses on genetically engineered mouse models, and in silico systems biology and bioinformatics analyses on CD151-related events. RESULTS: Endothelial ablation of Cd151 leads to pulmonary and cardiac inflammation, severe sepsis, and perilous COVID-19, and endothelial CD151 becomes downregulated in inflammation. Mechanistically, CD151 restrains endothelial release of proinflammatory molecules for less leukocyte infiltration. At the subcellular level, CD151 determines the integrity of multivesicular bodies/lysosomes and confines the production of exosomes that carry cytokines such as ANGPT2 (angiopoietin-2) and proteases such as cathepsin-D. At the molecular level, CD151 docks VCP (valosin-containing protein)/p97, which controls protein quality via mediating deubiquitination for proteolytic degradation, onto endolysosomes to facilitate VCP/p97 function. At the endolysosome membrane, CD151 links VCP/p97 to (1) IFITM3 (interferon-induced transmembrane protein 3), which regulates multivesicular body functions, to restrain IFITM3-mediated exosomal sorting, and (2) V-ATPase, which dictates endolysosome pH, to support functional assembly of V-ATPase. CONCLUSIONS: Distinct from its canonical function in strengthening cell adhesion at cell surface, CD151 maintains endolysosome function by sustaining VCP/p97-mediated protein unfolding and turnover. By supporting protein quality control and protein degradation, CD151 prevents proteins from (1) buildup in endolysosomes and (2) discharge through exosomes, to limit vascular inflammation. Also, our study conceptualizes that balance between degradation and discharge of proteins in endothelial cells determines vascular information. Thus, the IFITM3/V-ATPase-tetraspanin-VCP/p97 complexes on endolysosome, as a protein quality control and inflammation-inhibitory machinery, could be beneficial for therapeutic intervention against vascular inflammation.

Atherosclerotic burden and cerebral small vessel disease: exploring the link through microvascular aging and cerebral microhemorrhages.

Cerebral microhemorrhages (CMHs, also known as cerebral microbleeds) are a critical but frequently underestimated aspect of cerebral small vessel disease (CSVD), bearing substantial clinical consequences. Detectable through sensitive neuroimaging techniques, CMHs reveal an extensive pathological landscape. They are prevalent in the aging population, with multiple CMHs often being observed in a given individual. CMHs are closely associated with accelerated cognitive decline and are increasingly recognized as key contributors to the pathogenesis of vascular cognitive impairment and dementia (VCID) and Alzheimer's disease (AD). This review paper delves into the hypothesis that atherosclerosis, a prevalent age-related large vessel disease, extends its pathological influence into the cerebral microcirculation, thereby contributing to the development and progression of CSVD, with a specific focus on CMHs. We explore the concept of vascular aging as a continuum, bridging macrovascular pathologies like atherosclerosis with microvascular abnormalities characteristic of CSVD. We posit that the same risk factors precipitating accelerated aging in large vessels (i.e., atherogenesis), primarily through oxidative stress and inflammatory pathways, similarly instigate accelerated microvascular aging. Accelerated microvascular aging leads to increased microvascular fragility, which in turn predisposes to the formation of CMHs. The presence of hypertension and amyloid pathology further intensifies this process. We comprehensively overview the current body of evidence supporting this interconnected vascular hypothesis. Our review includes an examination of epidemiological data, which provides insights into the prevalence and impact of CMHs in the context of atherosclerosis and CSVD. Furthermore, we explore the shared mechanisms between large vessel aging, atherogenesis, microvascular aging, and CSVD, particularly focusing on how these intertwined processes contribute to the genesis of CMHs. By highlighting the role of vascular aging in the pathophysiology of CMHs, this review seeks to enhance the understanding of CSVD and its links to systemic vascular disorders. Our aim is to provide insights that could inform future therapeutic approaches and research directions in the realm of neurovascular health.

[Corrigendum] The tetraspanin CD151­ARSA mutant inhibits angiogenesis via the YRSL sequence.

Subsequently to the publication of the above paper, an interested reader drew to the authors' attention that, in Fig. 4A on p. 839, the 'CD151/24 h' and 'CD151­ARSA/48 h' panels appeared to contain overlapping sections of data, such that they were potentially derived from the same original source, where these panels were intended to show the results from differently performed experiments. The authors have re­examined their original data, and realize that the 'CD151­ARSA/48 h' panel was inadvertently placed incorrectly in the figure. The revised version of Fig. 4, now containing the correct data for the 'CD151­ARSA/48 h' experiment in Fig. 4A, is shown below. Note that this error did not adversely affect either the results or the overall conclusions reported in this study. All the authors agree with the publication of this corrigendum, and are grateful to the Editor of Molecular Medicine Reports for allowing them the opportunity to publish this. They also wish to apologize to the readership of the Journal for any inconvenience caused. [Molecular Medicine Reports 7: 836­842, 2013; DOI: 10.3892/mmr.2012.1250].

Tetraspanin Tspan8 restrains interferon signaling to stabilize intestinal epithelium by directing endocytosis of interferon receptor.

Inflammation can impair intestinal barrier, while increased epithelial permeability can lead to inflammation. In this study, we found that the expression of Tspan8, a tetraspanin expressed specifically in epithelial cells, is downregulated in mouse model of ulcerative disease (UC) but correlated with those of cell-cell junction components, such as claudins and E-cadherin, suggesting that Tspan8 supports intestinal epithelial barrier. Tspan8 removal increases intestinal epithelial permeability and upregulates IFN-γ-Stat1 signaling. We also demonstrated that Tspan8 coalesces with lipid rafts and facilitates IFNγ-R1 localization at or near lipid rafts. As IFN-γ induces its receptor undergoing clathrin- or lipid raft-dependent endocytosis and IFN-γR endocytosis plays an important role in Jak-Stat1 signaling, our analysis on IFN-γR endocytosis revealed that Tspan8 silencing impairs lipid raft-mediated but promotes clathrin-mediated endocytosis of IFN-γR1, leading to increased Stat1 signaling. These changes in IFN-γR1 endocytosis upon Tspan8 silencing correlates with fewer lipid raft component GM1 at the cell surface and more clathrin heavy chain in the cells. Our findings indicate that Tspan8 determines the IFN-γR1 endocytosis route, to restrain Stat1 signaling, stabilize intestine epithelium, and subsequently prevent intestine from inflammation. Our finding also implies that Tspan8 is needed for proper endocytosis through lipid rafts.

EWI2 and its relatives in Tetraspanin-enriched membrane domains regulate malignancy.

Experimental studies on immunoglobulin superfamily (IgSF) member EWI2 reveal that it suppresses a variety of solid malignant tumors including brain, lung, skin, and prostate cancers in animal models and inhibits tumor cell movement and growth in vitro. While EWI2 appears to support myeloid leukemia in mouse models and maintain leukemia stem cells. Bioinformatics analyses suggest that EWI2 gene expression is downregulated in glioblastoma but upregulated in melanoma, pancreatic cancer, and liver cancer. The mechanism of action for EWI2 is linked to its inhibition of growth factor receptors and cell adhesion proteins through its associated tetraspanin-enriched membrane domains (TEMDs), by altering the cell surface clustering and endolysosome trafficking/turnover of these transmembrane proteins. Recent studies also show that EWI2 modulates the nuclear translocation of ERK and TFEB to change the activities of these gene expression regulators. For EWI2 relatives including FPRP, IgSF3, and CD101, although their roles in malignant diseases are not fully clear and remain to be determined experimentally, FPRP and IgSF3 likely promote the progression of solid malignant tumors while CD101 seems to modulate immune cells of tumor microenvironment. Distinctive from other tumor regulators, the impacts of EWI subfamily members on solid malignant tumors are likely to be context dependent. In other words, the effect of a given EWI subfamily member on a tumor probably depends on the molecular network and composition of TEMDs in that tumor. Collectively, EWI2 and its relatives are emerged as important regulators of malignant diseases with promising potentials to become anti-cancer therapeutics and cancer therapy targets.

P66Shc (Shc1) Zebrafish Mutant Line as a Platform for Testing Decreased Reactive Oxygen Species in Pathology.

Reactive oxygen species (ROS) dysregulation exacerbates many pathologies but must remain within normal ranges to maintain cell function. Since ROS-mediated pathology and routine cell function are coupled, in vivo models evaluating low-ROS background effects on pathology are limited. Some models alter enzymatic antioxidant expression/activity, while others involve small molecule antioxidant administration. These models cause non-specific ROS neutralization, decreasing both beneficial and detrimental ROS. This is detrimental in cardiovascular pathology, despite the negative effects excessive ROS has on these pathologies. Thus, current trends in ROS-mediated pathology have shifted toward selective inhibition of ROS producers that are dysregulated during pathological insults, such as p66Shc. In this study, we evaluated a zebrafish heterozygote p66Shc hypomorphic mutant line as a low-ROS myocardial infarction (MI) pathology model that mimics mammalian MI. Our findings suggest this zebrafish line does not have an associated negative phenotype, but has decreased body mass and tissue ROS levels that confer protection against ROS-mediated pathology. Therefore, this line may provide a low-ROS background leading to new insights into disease.

Overproduction of Membrane-Associated, and Integrated, Proteins Using Saccharomyces cerevisiae.

Structural and functional eukaryotic membrane protein research continues to grow at an increasing rate, placing greater significance on leveraging productive protein expression pipelines to feed downstream studies. Bacterial expression systems (e.g., E. coli) are often the preferred system due to their simple growth conditions, relative simplicity in experimental workflow, low overall cost per liter of cell growth, and ease of genetic manipulation. However, overproduction success of eukaryotic membrane proteins in bacterial systems is hindered by the limited native processing ability of bacterial systems for important protein folding interactions (e.g., disulfide bonds), post-translational modifications (e.g., glycosylation), and inherent disadvantages in protein trafficking and folding machinery compared to other expression systems.In contrast, Saccharomyces cerevisiae expression systems combine positive benefits of simpler bacterial systems with those of more complex eukaryotic systems (e.g., mammalian cells). Benefits include inexpensive growth, robust DNA repair and recombination machinery, amenability to high density growths in bioreactors, efficient transformation, and robust post-translational modification machinery. These characteristics make S. cerevisiae a viable first-alternative when bacterial overproduction is insufficient. Thus, this chapter provides a framework, using methods that have proven successful in prior efforts, for overproducing membrane anchored or membrane integrated proteins in S. cerevisiae. The framework is designed to improve yields for all levels of overexpression expertise, providing optimization insights for the variety of processes involved in heterologous protein expression.

Purification of Membrane Proteins Overexpressed in Saccharomyces cerevisiae.

Membrane protein (MP) functional and structural characterization requires large quantities of high-purity protein for downstream studies. Barriers to MP characterization include ample overexpression, solubilization, and purification of target proteins while maintaining native activity and structure. These barriers can be overcome by utilizing an efficient purification protocol in a high-yield eukaryotic expression system such as Saccharomyces cerevisiae. S. cerevisiae offers improved protein folding and posttranslational modifications compared to prokaryotic expression systems. This chapter contains practices used to overcome barriers of solubilization and purification using S. cerevisiae that are broadly applicable to diverse membrane associated, and membrane integrated, protein targets.

EWI2 prevents EGFR from clustering and endocytosis to reduce tumor cell movement and proliferation.

EWI2 is a transmembrane immunoglobulin superfamily (IgSF) protein that physically associates with tetraspanins and integrins. It inhibits cancer cells by influencing the interactions among membrane molecules including the tetraspanins and integrins. The present study revealed that, upon EWI2 silencing or ablation, the elevated movement and proliferation of cancer cells in vitro and increased cancer metastatic potential and malignancy in vivo are associated with (i) increases in clustering, endocytosis, and then activation of EGFR and (ii) enhancement of Erk MAP kinase signaling. These changes in signaling make cancer cells (i) undergo partial epithelial-to-mesenchymal (EMT) for more tumor progression and (ii) proliferate faster for better tumor formation. Inhibition of EGFR or Erk kinase can abrogate the cancer cell phenotypes resulting from EWI2 removal. Thus, to inhibit cancer cells, EWI2 prevents EGFR from clustering and endocytosis to restrain its activation and signaling.

EWI2 promotes endolysosome-mediated turnover of growth factor receptors and integrins to suppress lung cancer.

As a partner of tetraspanins, EWI2 suppresses glioblastoma, melanoma, and prostate cancer; but its role in lung cancer has not been investigated. Bioinformatics analysis reveals that EWI2 gene expression is up regulated in lung adenocarcinoma and higher expression of EWI2 mRNA may predict poorer overall survival. However, experimental analysis shows that EWI2 protein is actually downregulated constantly in the tissues of lung adenocarcinoma and lung squamous cell carcinoma. Forced expression of EWI2 in human lung adenocarcinoma cells reduces total cellular and cell surface levels of various integrins and growth factor receptors, which initiates the outside-in motogenic and mitogenic signaling. These reductions result in the decreases in 1) cell-matrix adhesion, cell movement, and cell transformation in vitro and 2) tumor growth, burden, and metastasis in vivo, and result from the increases in lysosomal trafficking and proteolytic degradation of theses membrane receptors. EWI2 elevates lysosome formation by promoting nuclear retention of TFEB, the master transcription factor driving lysosomogenesis. In conclusion, EWI2 as a lung cancer suppressor attenuates lung cancer cells in a comprehensive fashion by inhibiting both tumor growth and tumor metastasis; EWI2 as an endolysosome regulator promotes lysosome activity to enhance lysosomal degradation of growth factor receptors and integrins and then reduce their levels and functions; and EWI2 can become a promising therapeutic candidate given its accessibility at the cell surface, dual inhibition on growth factor receptors and integrins, and broad-spectrum anti-cancer activity. More importantly, our observations also provide a novel therapeutic strategy to bypass the resistance to EGFR inhibitors.

Cardiac Involvement in Recovered Patients From COVID-19: A Preliminary 6-Month Follow-Up Study.

Background: Accumulating evidence has revealed that coronavirus disease 2019 (COVID-19) patients may be complicated with myocardial injury during hospitalization. However, data regarding persistent cardiac involvement in patients who recovered from COVID-19 are limited. Our goal is to further explore the sustained impact of COVID-19 during follow-up, focusing on the cardiac involvement in the recovered patients. Methods: In this prospective observational follow-up study, we enrolled a total of 40 COVID-19 patients (20 with and 20 without cardiac injury during hospitalization) who were discharged from Zhongnan Hospital of Wuhan University for more than 6 months, and 27 patients (13 with and 14 without cardiac injury during hospitalization) were finally included in the analysis. Clinical information including self-reported symptoms, medications, laboratory findings, Short Form 36-item scores, 6-min walk test, clinical events, electrocardiogram assessment, echocardiography measurement, and cardiac magnetic resonance imaging was collected and analyzed. Results: Among 27 patients finally included, none of patients reported any obvious cardiopulmonary symptoms at the 6-month follow-up. There were no statistically significant differences in terms of the quality of life and exercise capacity between the patients with and without cardiac injury. No significant abnormalities were detected in electrocardiogram manifestations in both groups, except for nonspecific ST-T changes, premature beats, sinus tachycardia/bradycardia, PR interval prolongation, and bundle-branch block. All patients showed normal cardiac structure and function, without any statistical differences between patients with and without cardiac injury by echocardiography. Compared with patients without cardiac injury, patients with cardiac injury exhibited a significantly higher positive proportion in late gadolinium enhancement sequences [7/13 (53.8%) vs. 1/14 (7.1%), p = 0.013], accompanied by the elevation of circulating ST2 level [median (interquartile range) = 16.6 (12.1, 22.5) vs. 12.5 (9.5, 16.7); p = 0.044]. Patients with cardiac injury presented higher levels of aspartate aminotransferase, creatinine, high-sensitivity troponin I, lactate dehydrogenase, and N-terminal pro-B-type natriuretic peptide than those without cardiac injury, although these indexes were within the normal range for all recovered patients at the 6-month follow-up. Among patients with cardiac injury, patients with positive late gadolinium enhancement presented higher cardiac biomarker (high-sensitivity troponin I) and inflammatory factor (high-sensitivity C-reactive protein) on admission than the late gadolinium enhancement-negative subgroup. Conclusions: Our preliminary 6-month follow-up study with a limited number of patients revealed persistent cardiac involvement in 29.6% (8/27) of recovered patients from COVID-19 after discharge. Patients with cardiac injury during hospitalization were more prone to develop cardiac fibrosis during their recovery. Among patients with cardiac injury, those with relatively higher cardiac biomarkers and inflammatory factors on admission appeared more likely to have cardiac involvement in the convalescence phase.

A Bioinformatics Perspective on the Links Between Tetraspanin-Enriched Microdomains and Cardiovascular Pathophysiology.

Background: Tetraspanins and integrins are integral membrane proteins. Tetraspanins interact with integrins to modulate the dynamics of adhesion, migration, proliferation, and signaling in the form of membrane domains called tetraspanin-enriched microdomains (TEMs). TEMs also contain other cell adhesion proteins like immunoglobulin superfamily (IgSF) proteins and claudins. Cardiovascular functions of these TEM proteins have emerged and remain to be further revealed. Objectives: The aims of this study are to explore the roles of these TEM proteins in the cardiovascular system using bioinformatics tools and databases and to highlight the TEM proteins that may functionally associate with cardiovascular physiology and pathology. Methods: For human samples, three databases-GTEx, NCBI-dbGaP, and NCBI-GEO-were used for the analyses. The dbGaP database was used for GWAS analysis to determine the association between target genes and human phenotypes. GEO is an NCBI public repository that archives genomics data. GTEx was used for the analyses of tissue-specific mRNA expression levels and eQTL. For murine samples, GeneNetwork was used to find gene-phenotype correlations and gene-gene correlations of expression levels in mice. The analysis of cardiovascular data was the focus of this study. Results: Some integrins and tetraspanins, such as ITGA8 and Cd151, are highly expressed in the human cardiovascular system. TEM components are associated with multiple cardiovascular pathophysiological events in humans. GWAS and GEO analyses showed that human Cd82 and ITGA9 are associated with blood pressure. Data from mice also suggest that various cardiovascular phenotypes are correlated with integrins and tetraspanins. For instance, Cd82 and ITGA9, again, have correlations with blood pressure in mice. Conclusion: ITGA9 is related to blood pressure in both species. KEGG analysis also linked ITGA9 to metabolism and MAPK signaling pathway. This work provides an example of using integrated bioinformatics approaches across different species to identify the connections of structurally and/or functionally related molecules to certain categories of diseases.

Endothelial dysfunction in COVID-19 calls for immediate attention: the emerging roles of the endothelium in inflammation caused by SARS-CoV-2.

The COVID-19 pandemic has caused numerous deaths around the world. A growing body of evidence points to the important role of overwhelming inflammatory responses in the pathogenesis of COVID-19 and the effectiveness of anti-inflammation therapy against COVID-19 is emerging. In addition to affecting the lungs, COVID-19 can be a severe systemic inflammatory disease that is related to endothelial dysfunction. We are calling for closer attention to endothelial dysfunction in COVID-19 not only for fully revealing the pathogenic mechanism of COVID-19 but also for properly adjusting the strategy of clinical intervention.

Association of Statin Use With the In-Hospital Outcomes of 2019-Coronavirus Disease Patients: A Retrospective Study.

Background: Statins have multiple protective effects on inflammation, immunity and coagulation, and may help alleviate pneumonia. However, there was no report focusing on the association of statin use with in-hospital outcomes of patients with coronavirus disease 2019 (COVID-19). We investigated the association between the use of statins and in-hospital outcomes of patients with COVID-19. Methods: In this retrospective case series, consecutive COVID-19 patients admitted at 2 hospitals in Wuhan, China, from March 12, 2020 to April 14, 2020 were analyzed. A 1:1 matched cohort was created by propensity score-matched analysis. Demographic data, laboratory findings, comorbidities, treatments and in-hospital outcomes were collected and compared between COVID-19 patients taking and not taking statins. Result: A total of 2,147 patients with COVID-19 were enrolled in this study. Of which, 250 patients were on statin therapy. The mortality was 2.4% (6/250) for patients taking statins while 3.7% (70/1,897) for those not taking statins. In the multivariate Cox model, after adjusting for age, gender, admitted hospital, comorbidities, in-hospital medications and blood lipids, the risk was lower for mortality (adjusted HR, 0.428; 95% CI, 0.169-0.907; P = 0.029), acute respiratory distress syndrome (ARDS) (adjusted HR, 0.371; 95% CI, 0.180-0.772; P = 0.008) or intensive care unit (ICU) care (adjusted HR, 0.319; 95% CI, 0.270-0.945; P = 0.032) in the statin group vs. the non-statin group. After propensity score-matched analysis based on 18 potential confounders, a 1:1 matched cohort (206:206) was created. In the matched cohort, the Kaplan-Meier survival curves showed that the use of statins was associated with better survival (P = 0.025). In a Cox regression model, the use of statins was associated with lower risk of mortality (unadjusted HR, 0.254; 95% CI, 0.070-0.926; P = 0.038), development of ARDS (unadjusted HR, 0.240; 95% CI, 0.087-0.657; P = 0.006), and admission of ICU (unadjusted HR, 0.349; 95% CI, 0.150-0.813; P = 0.015). The results remained consistent when being adjusted for age, gender, total cholesterol, triglyceride, low density lipoprotein cholesterol, procalcitonin, and brain natriuretic peptide. The favorable outcomes in statin users remained statistically significant in the first sensitivity analysis with comorbid diabetes being excluded in matching and in the second sensitivity analysis with chronic obstructive pulmonary disease being added in matching. Conclusion: In this retrospective analysis, the use of statins in COVID-19 patients was associated with better clinical outcomes and is recommended to be continued in patients with COVID-19.

Decreased serum albumin level indicates poor prognosis of COVID-19 patients: hepatic injury analysis from 2,623 hospitalized cases.

Coronavirus disease 2019 (COVID-19) is a global pandemic which has caused numerous deaths worldwide. The present study investigated the roles of hypoproteinemia in the clinical outcome and liver dysfunction of COVID-19 patients. In this retrospective study, we extracted data from 2,623 clinically confirmed adult COVID-19 patients (>18 years old) between January 29, 2020 and March 6, 2020 in Tongji Hospital, Wuhan, China. The patients were divided into three groups-non-critically ill, critically ill, and death groups-in accordance with the Chinese Clinical Guideline for COVID-19. Serum albumin, low-density lipoproteins cholesterol (LDL-C), and high-density lipoproteins cholesterol (HDL-C) concentrations and inflammatory cytokines levels were measured and compared among these three groups. The median age of these 2,623 patients was 64 years old (interquartile range (IQR), 52-71). Among the patients enrolled in the study, 2,008 (76.6%) were diagnosed as non-critically ill and 615 (23.4%) were critically ill patients, including 383 (14.6%) critically ill survivors and 232 (8.8%) critically ill deaths in the hospital. Marked hypoalbuminemia occurred in 38.2%, 71.2%, and 82.4% patients in non-critically ill, critically ill, and death groups, respectively, on admission and 45.9%, 77.7%, and 95.6% of these three groups, respectively, during hospitalization. We also discovered that serum low-density lipoprotein (LDL) and HDL levels were significantly lower in critically ill and death groups compared to non-critically ill group. Meanwhile, the patients displayed dramatically elevated levels of serum inflammatory factors, while a markedly prolonged activated partial thromboplastin time (APTT) in critically ill patients reflected coagulopathy. This study suggests that COVID-19-induced cytokine storm causes hepatotoxicity and subsequently critical hypoalbuminemia, which are associated with exacerbation of disease-associated inflammatory responses and progression of the disease and ultimately leads to death for some critically ill patients.

A novel role of LRP5 in tubulointerstitial fibrosis through activating TGF-ß/Smad signaling.

Previous studies by us and others demonstrated that activation of Wnt/ß-catenin signaling plays a pathogenic role in chronic kidney diseases (CKD). Wnt co-receptor LRP5 variants are reported to associate with autosomal dominant polycystic kidney disease; but their exact roles in this disease and renal fibrosis have not been explored. Here, we observed the upregulation of LRP5 in the renal tubules of both type 1 and type 2 diabetic models and of an obstructive nephropathy model. In the obstructed kidneys, Lrp5 knockout significantly ameliorated tubulointerstitial fibrosis and tubular injury without changing Wnt/ß-catenin signaling. Instead, decreased levels of TGF-ß1 and TGF-ß receptors (TßRs) were detected in Lrp5 knockout kidneys, followed by attenuated activation and nuclear translocation of Smad2/3 in the renal tubules, suggesting a regulatory effect of LRP5 on TGF-ß/Smad signaling. In consistent with this hypothesis, LRP5 overexpression resulted in enhanced TGF-ß/Smad signaling activation in renal tubule epithelial cells. Furthermore, LRP5 was co-immunoprecipitated with TßRI and TßRII, and its extracellular domain was essential for interacting with TßRs and for its pro-fibrotic activity. In addition to stabilizing TßRs, LRP5 increased the basal membrane presentation and TGF-ß1-induced internalization of these receptors. Notably, TGF-ß1 also induced LRP5 internalization. These findings indicate that LRP5 promotes tubulointerstitial fibrosis, at least partially, via direct modulation of TGF-ß/Smad signaling, a novel, Wnt-independent function.

CD82-TRPM7-Numb signaling mediates age-related cognitive impairment.

Aging is a crucial cause of cognitive decline and a major risk factor for Alzheimer's disease (AD); however, AD's underlying molecular mechanisms remain unclear. Recently, tetraspanins have emerged as important modulators of synaptic function and memory. We demonstrate that the level of tetraspanin CD82 is upregulated in the brains of AD patients and middle-aged mice. In young adult mice, injection of AAV-CD82 to the hippocampus induced AD-like cognitive deficits and impairments in neuronal spine density. CD82 overexpression increased TRPM7 α-kinase cleavage via caspase-3 activation and induced Numb phosphorylation at Thr346 and Ser348 residues. CD82 overexpression promoted beta-amyloid peptide (Aß) secretion which could be reversed by Numb T346S348 mutants. Importantly, hippocampus-related memory functions were improved in Cd82-/- mice. Taken together, our findings provide the evidence that links the elevated CD82-TRPM7-Numb signaling to age-related cognitive impairment.

Tetraspanin CD82 interaction with cholesterol promotes extracellular vesicle-mediated release of ezrin to inhibit tumour cell movement.

Tumour metastasis suppressor KAI1/CD82 inhibits tumour cell movement. As a transmembrane protein, tetraspanin CD82 bridges the interactions between membrane microdomains of lipid rafts and tetraspanin-enriched microdomains (TEMs). In this study, we found that CD82 and other tetraspanins contain cholesterol recognition/interaction amino-acid consensus (CRAC) sequences in their transmembrane domains and revealed that cholesterol binding of CD82 determines its interaction with lipid rafts but not with TEMs. Functionally, CD82 needs cholesterol binding to inhibit solitary migration, collective migration, invasion and infiltrative outgrowth of tumour cells. Importantly, CD82-cholesterol/-lipid raft interaction not only promotes extracellular release of lipid raft components such as cholesterol and gangliosides but also facilitates extracellular vesicle (EV)-mediated release of ezrin-radixin-moesin (ERM) protein Ezrin. Since ERM proteins link actin cytoskeleton to the plasma membrane, we show for the first time that cell movement can be regulated by EV-mediated releases, which disengage the plasma membrane from cytoskeleton and then impair cell movement. Our findings also conceptualize that interactions between membrane domains, in this case converge of lipid rafts and TEMs by CD82, can change cell movement. Moreover, CD82 coalescences with both lipid rafts and TEMs are essential for its inhibition of tumour cell movement and for its enhancement of EV release. Finally, our study underpins that tetraspanins as a superfamily of functionally versatile molecules are cholesterol-binding proteins. Abbreviations: Ab: antibody; CBM: cholesterol-binding motif; CCM: cholesterol consensus motif; CRAC/CARC: cholesterol recognition or interaction amino-acid consensus; CTxB: cholera toxin B subunit; ECM: extracellular matrix; ERM: ezrin, radixin and moesin; EV: extracellular vesicles; FBS: foetal bovine serum; mAb: monoclonal antibody; MST: microscale thermophoresis; pAb: polyclonal antibody; and TEM: tetraspanin-enriched microdomain.

Nicotinamide mononucleotide (NMN) treatment attenuates oxidative stress and rescues angiogenic capacity in aged cerebromicrovascular endothelial cells: a potential mechanism for the prevention of vascular cognitive impairment.

Age-related impairment of angiogenesis likely has a critical role in cerebromicrovascular rarefaction and development of vascular cognitive impairment and dementia (VCID) in the elderly. Recently, we demonstrated that aging is associated with NAD+ depletion in the vasculature and that administration of NAD+ precursors exerts potent anti-aging vascular effects, rescuing endothelium-mediated vasodilation in the cerebral circulation and improving cerebral blood supply. The present study was designed to elucidate how treatment with nicotinamide mononucleotide (NMN), a key NAD+ intermediate, impacts age-related impairment of endothelial angiogenic processes. Using cerebromicrovascular endothelial cells (CMVECs) isolated from young and aged F344xBN rats, we demonstrated that compared with young cells, aged CMVECs exhibit impaired proliferation, cellular migration (measured by a wound-healing assay using electric cell-substrate impedance sensing [ECIS] technology), impaired ability to form capillary-like structures, and increased oxidative stress. NMN treatment in aged CMVECs significantly improved angiogenic processes and attenuated H2O2 production. We also found that pre-treatment with EX-527, a pharmacological inhibitor of SIRT1, prevented NMN-mediated restoration of angiogenic processes in aged CMVECs. Collectively, we find that normal cellular NAD+ levels are essential for normal endothelial angiogenic processes, suggesting that age-related cellular NAD+ depletion and consequential SIRT1 dysregulation may be a potentially reversible mechanism underlying impaired angiogenesis and cerebromicrovascular rarefaction in aging. We recommend that pro-angiogenic effects of NAD+ boosters should be considered in both preclinical and clinical studies.