Inhibition of the CoREST Repressor Complex Promotes Wound Re-Epithelialization through the Regulation of Keratinocyte Migration.

Wound healing is a complex process involving phases of hemostasis, inflammation, proliferation, and remodeling. The regenerative process in the skin requires coordination between many regulators, including signaling molecules, transcription factors, and the epigenetic machinery. In this study, we show that chromatin regulators HDAC1 and LSD1, key components of the CoREST repressor complex, are upregulated in the regenerating epidermis during wound repair. We also show that corin, a synthetic dual inhibitor of the CoREST complex and HDAC1/LSD1 activities, significantly accelerates wound closure through enhanced re-epithelialization in a mouse tail wound model. Acetylated H3K9 (methylation of histone H3 at lysine 9) expression, a histone modification targeted by HDAC1, is increased in keratinocytes after topical treatment with 100 nM and 1 µM of corin. In vitro experiments demonstrate that corin promotes migration and inhibits the proliferation of human keratinocytes. Furthermore, expression levels of genes promoting keratinocyte migration, such as AREG, CD24, EPHB2, ITGAX, PTGS, SCT1, SERPINB2, SERPINE1, SLPI, SNAI2, and TWIST, increased in keratinocytes treated with corin. These data demonstrate that dual inhibition of class I histone deacetylases and LSD1 by corin may serve as a new approach for promoting wound re-epithelialization and provide a platform for further applications of corin for the treatment of chronic wounds.

Utility of human epididymis protein 4 in the differential diagnosis of ascites.

BACKGROUND AND AIMS: Human epididymal protein 4 (HE4) may be a useful tool in the differential diagnosis of malignant ascites. The aim of this study was to evaluate the diagnostic utility of HE4 for detecting malignant ascites, taking into account the possible false positives identified with adenosine deaminase (ADA), C-reactive protein (CRP), % polynuclear cells (%PMN) and glomerular filtration rate (eGFR). METHODS: Concentrations of HE4, ADA, %PMN and CRP were determined in 114 samples of peritoneal fluid and creatinine in serum in order to calculate eGFR. RESULTS: Concentrations of HE4 presented significant differences (P = 0.028) in benign [median (interquartile range)] [582(372)] pmol/L) and malignant ascites ([8241(367)] pmol/L. Sensitivity was 21.2% and specificity 100%. Significant differences were also observed for HE4 between tumors of gynecological origin ([3165(8769)] pmol/L) and others ([665(663)] pmol/L), with a sensitivity of 67% and a specificity of 100%. Classifying according to possible false positives (ADA > 45U/L, CRP > 50 mg/L, %PMN > 90 and eGFR < 30 mL/min/1.73 m2) at maximum specificity, a sensitivity of 33.3% was obtained for HE4, with a cut-off point of 2660 pmol/L. Without possible false positives (ADA < 45U/L, CRP < 50 mg/L, %PMN < 90 and eGFR ≥ 30 mL/min/1.73 m2), a sensitivity of 37.7% was obtained at 100% specificity for a cut-off point of 1041 pmol/L. Applying these criteria to the entire group, a sensitivity of 36.4% was obtained at maximum specificity. CONCLUSIONS: HE4 allows the identification of malignant ascites with moderate sensitivity at maximum specificity. HE4 levels can differentiate between tumors of gynecological origin and others. Classification according to possible false positives increases sensitivity without losing specificity.

Medication Assessment in an Older Population during Acute Care Hospitalization and Its Effect on the Anticholinergic Burden: A Prospective Cohort Study.

(1) Background: Anticholinergic and sedative drugs (ASDs) contribute to negative health outcomes, especially in the frail population. In this study, we aimed to assess whether frailty increases with anticholinergic burden and to evaluate the effects of medication reviews (MRs) on ASD regimens among patients attending an acute care for the elderly (ACE) unit. (2) Methods: A cohort study was conducted between June 2019 and October 2020 with 150 consecutive patients admitted to our ACE unit. Demographic, clinical, and pharmacological data were assessed. Frailty score was determined using the Frail-VIG index (FI-VIG), and ASD burden was quantified using the drug burden index (DBI). In addition, the MR was performed using the patient-centered prescription (PCP) model. We used a paired T-test to compare the DBI pre- and post-MR and univariate and multivariate regression to identify the factors associated with frailty. (3) Results: Overall, 85.6% (n = 128) of participants showed some degree of frailty (FI-VIG > 0.20) and 84% (n = 126) of patients received treatment with ASDs upon admission (pre-MR). As the degree of frailty increased, so did the DBI (p < 0.001). After the implementation of the MR through the application of the PCP model, a reduction in the DBI was noted (1.06 ± 0.8 versus 0.95 ± 0.7) (p < 0.001). After adjusting for covariates, the association between frailty and the DBI was apparent (OR: 11.42, 95% (CI: 2.77-47.15)). (4) Conclusions: A higher DBI was positively associated with frailty. The DBI decreased significantly in frail patients after a personalized MR. Thus, MRs focusing on ASDs are crucial for frail older patients.

Abordaje multidisciplinarde un paciente con una úlceraperianal asociada a pomadaantihemorroidal / Multidisciplinary approach to a patient with a perianal ulcer associated with hemorrhoidal ointment

Introducción: Hasta la fecha, la manifestación de una úlcera perianalprovocada por una pomada antihemorroidal no se ha descrito confrecuencia. Sin embargo, se ha objetivado un incremento de loscasos durante la pandemia de COVID-19. Caso clínico: Varónde 82 años independiente, que presentó una úlcera perianal de35,8 cm² sin ninguna patología ni enfermedad concomitante queexplicara su causa. La aplicación de criterios de exclusión exhaustivos,incluida una biopsia para rechazar el pioderma gangrenoso,identificó una pomada rectal hemorroidal como la causa de la úlcera.Plan de actuación: La herida curó tras aplicar una intervenciónmultidisciplinaria y una terapia con factores de crecimientoautólogos. Discusión y conclusiones: Este caso ha sido escasamentereportado en la literatura, aunque esta pomada hemorroidal secomercializa desde hace más de 40 años. Se recomienda evaluaciónmédica antes de la prescripción. (AU)

Introduction: Perianal ulcers resulting from the use of hemorrhoidalointments have been rarely reported to date. Nevertheless, therehas been a surge in the number of cases reported during theCOVID-19 pandemic. Case report: An independent 82-year-oldmale experienced a 35,80 cm² perianal ulcer, with no underlyingcondition or concomitant disease that could explain the cause ofthe ulcer. The application of thorough exclusion criteria, including abiopsy to rule out pyoderma gangrenosum, led to the identificationof a hemorrhoidal rectal ointment as the cause. Action plan: Theulcer healed completely when a multidisciplinary intervention and anautologous growth factors advanced therapy were applied. Discussionand conclusions: This case has been scarcely reported in the literature,although this hemorrhoidal ointment has been on the market for over40 years. Medical assessment before prescription and patients’ followup is recommended. (AU)

Autologous Cultured Bone Marrow-Derived Mesenchymal Stem Cells in a Fibrin Spray to Treat Venous Ulcers: A Randomized Controlled Double-Blind Pilot Study.

We treated a small cohort of venous ulcers that were very unresponsive to standard and advanced therapies with autologous cultured bone marrow-derived mesenchymal stem cells (MSCs). This pilot clinical trial was randomized, controlled, and double-blinded. Subjects were treated with either normal saline (Group A), fibrin spray alone (Group B), or MSCs in fibrin (1 million cells/cm2 of wound bed surface) (Group C). The control and test materials were applied to the wound using a double-barreled syringe with thrombin and fibrinogen (with or without MSCs) in each barrel, or saline alone in both barrels. The MSCs were separated, cultured in vitro, and expanded in a dedicated Good Manufacturing Practice (GMP) facility from 30-50 ml of bone marrow aspirate obtained from the iliac crest in Group C subjects. To ensure that the study remained controlled and blinded, subjects who were randomized to one of the two control arms (saline or fibrin) underwent sham bone marrow aspiration performed by a hematologist who anesthetized the iliac crest area down to and pushing against the periosteum, but without penetrating the bone marrow. Therefore, both the clinician who evaluated wound progress and the study subjects had no knowledge of whether bone aspiration was actually performed and what treatment had been applied to the wound. The study was performed after full FDA investigational new drug (IND) approval. The primary endpoint was the rate of healing (wound closure as linear healing from the wound margins in cm/week), as measured by the Gilman equation. One-way ANOVA was used to calculate the statistical significance of differences between the mean healing rates of each of the 3 treatment groups every 4 weeks and over the 24 weeks of treatment. Overall, treatment with MSCs accelerated the healing rate by about 10-fold compared to those in the saline and fibrin control groups. Although the total number of patients in this pilot study was small (n=11), the statistical significance was surprisingly promising: p<0.01 and f-ratio of 15.9358. No serious adverse events were noted. This small but carefully performed prospective, controlled, randomized, and double-blinded pilot study in a rare population of totally unresponsive patients adds to previous reports showing the promise of MSCs in the treatment of chronic wounds and provides proof of principle for how to approach this type of very demanding clinical and translational research.

Assessment of frailty in elderly patients attending a multidisciplinary wound care centre: a cohort study.

BACKGROUND: The incidence of frailty and non-healing wounds increases with patients' age. Knowledge of the relationship between frailty and wound healing progress is greatly lacking. METHODS: The aim of this study is to characterize the degree of frailty in elderly patients attending a multidisciplinary wound care centres (MWCC). Additionally, we seek to assess the impact of frailty on the wound healing rate and wound healing time. An open cohort study was conducted on 51 consecutive patients aged > 70 years treated for wounds at an MWCC of an intermediate care hospital. The frailty score was determined according to the Frail-VIG index. Data were collected through patient questionnaires at the beginning of the study, and at 6 months or upon wound healing. Wounds were followed up every 2 weeks. To analyse the relationship between two variables was used the Chi-square test and Student's or the ANOVA model. The t-test for paired data was used to analyse the evolution of the frailty index during follow-up. RESULTS: A total of 51 consecutive patients were included (aged 81.1 ± 6.1 years). Frailty prevalence was 74.5% according to the Frail-VIG index (47.1% mildly frail, 19.6% moderately frail, and 7.8% severely frail). Wounds healed in 69.6% of cases at 6 months. The frailty index (FI) was higher in patients with non-healing wounds in comparison with patients with healing wounds (IF 0.31 ± 0.15 vs IF 0.24 ± 0.11, p = 0.043). A strong correlation between FI and wound healing results was observed in patients with non-venous ulcers (FI 0.37 ± 0.13 vs FI 0.27 ± 0.10, p = 0.015). However, no correlation was observed in patients with venous ulcers (FI 0.17 ± 0.09 vs FI 0.19 ± 0.09, p = 0.637). Wound healing rate is statically significantly higher in non-frail patients (8.9% wound reduction/day, P25-P75 3.34-18.3%/day;AQ6 p = 0.044) in comparison with frail patients (3.26% wound reduction/day, P25-P75 0.8-8.8%/day). CONCLUSION: Frailty is prevalent in elderly patients treated at an MWCC. Frailty degree is correlated with wound healing results and wound healing time.

Dermal Fibroblasts from Chronic Wounds Exhibit Paradoxically Enhanced Proliferative and Migratory Activities that May be Related to the Non-Canonical Wnt Signaling Pathway.

It is generally thought that dermal fibroblasts from chronic wounds are in a state of senescence, which contributes to the failure to heal. This assumption, based on limited experimental evidence, has led to the widespread use of therapeutic approaches focused on delivering new fibroblasts and/or increasing resident fibroblast activity to promote healing. In this study, we decided to re-visit the evidence for the relative inactivity of resident chronic wound fibroblasts. We therefore evaluated the proliferative and migratory activities of matching, patient-derived dermal fibroblasts from a chronic wound (wound dermal fibroblasts, or WDF), ipsilateral thigh newly created acute wound dermal fibroblasts (ADF, Day-3 after wounding the normal thigh skin), and ipsilateral thigh normal dermal skin fibroblasts (NDF). This approach was used in each of 10 consecutive non-selected individual patients with a venous leg ulcer, and allowed us to determine whether WDF are intrinsically less active than NDF and AWD. Cell migration and proliferation were quantified by a live-cell analysis system and MTT assay, respectively, in low (0.5%) or high (10%) levels of fetal bovine serum (FBS). In addition, the ability of patient-derived fibroblasts to modulate wound re-epithelialization in vivo was analyzed by transplantation in a mouse tail full-thickness wound model. Wnt5a mRNA, its ROR1 co-receptors, and ROR2 mRNA levels were determined by qRT-PCR. We report that WDF had increased -SMA and increased levels of Wnt5a. Moreover, using live-cell imaging in a scratch assay monolayer model, WDF showed baseline migratory activity similar to those of NDF and ADF, and such activity was not stimulated by FBS. WDF showed the same capacity to increase wound re-epithelialization as NDF and ADF. Together, these results suggest that WDF are not actually less "active" than NDF and ADF. This enhanced activity of chronic wound fibroblasts may lead to high energy requirements that contribute to a failure to heal. The findings may represent a new paradigm for wound chronicity, impaired healing, and high recurrence rates.

Convolutional Neural Network for Skin Lesion Classification: Understanding the Fundamentals Through Hands-On Learning.

Deep learning architectures for the classification of images have shown outstanding results in a variety of disciplines, including dermatology. The expectations generated by deep learning for, e.g., image-based diagnosis have created the need for non-experts to become familiar with the working principles of these algorithms. In our opinion, getting hands-on experience with these tools through a simplified but accurate model can facilitate their understanding in an intuitive way. The visualization of the results of the operations performed by deep learning algorithms on dermatological images can help students to grasp concepts like convolution, even without an advanced mathematical background. In addition, the possibility to tune hyperparameters and even to tweak computer code further empower the reach of an intuitive comprehension of these processes, without requiring advanced computational and theoretical skills. This is nowadays possible thanks to recent advances that have helped to lower technical and technological barriers associated with the use of these tools, making them accessible to a broader community. Therefore, we propose a hands-on pedagogical activity that dissects the procedures to train a convolutional neural network on a dataset containing images of skin lesions associated with different skin cancer categories. The activity is available open-source and its execution does not require the installation of software. We further provide a step-by-step description of the algorithm and of its functions, following the development of the building blocks of the computer code, guiding the reader through the execution of a realistic example, including the visualization and the evaluation of the results.

A toolkit for the quantitative evaluation of chronic wounds evolution for early detection of non-healing wounds.

BACKGROUND: Chronic wounds resulting from a number of conditions do not heal properly and can pose serious health problems. Beyond clinician visual inspection, an objective evaluation of the wound is required to assess wound evolution and the effectiveness of therapies. AIM: Our objective is to provide a methodology for the analysis of wound area vs. time for the early prediction of non-healing wounds evolution. METHODS: We propose a two-step approach consisting of: i) wound area quantification from planimetries and ii) classification of wound healing through the inference of characteristic parameters. For the first step, we describe a user-friendly software (Woundaries) to automatically calculate the wound area and other geometric parameters from hand-traced planimetries. For the second, we use a procedure for the objective classification of wound time evolution and the early assessment of treatment efficacy. The methodology was tested on simulations and retrospectively applied to data from 85 patients to compare the effect of a biological therapy with respect to general basic therapeutics. RESULTS: Woundaries provides measurements of wound surface equivalent to a validated device. The two-step methodology allows to determine if a wound is healing with high sensitivity, even with limited amount of data. Therefore, it allows the early assessment of the efficacy of a therapy. CONCLUSION: The performance of this methodology for the quantification and the objective evaluation of wound area evolution suggest it as a useful toolkit to assist clinicians in the early assessment of the efficacy of treatments, leading to a timely change of therapy.

Photoswitchable Antagonists for a Precise Spatiotemporal Control of ß2-Adrenoceptors.

ß2-Adrenoceptors (ß2-AR) are prototypical G-protein-coupled receptors and important pharmacological targets with relevant roles in physiological processes and diseases. Herein, we introduce Photoazolol-1-3, a series of photoswitchable azobenzene ß2-AR antagonists that can be reversibly controlled with light. These new photochromic ligands are designed following the azologization strategy, with a p-acetamido azobenzene substituting the hydrophobic moiety present in many ß2-AR antagonists. Using a fluorescence resonance energy transfer (FRET) biosensor-based assay, a variety of photopharmacological properties are identified. Two of the light-regulated molecules show potent ß2-AR antagonism and enable a reversible and dynamic control of cellular receptor activity with light. Their photopharmacological properties are opposite, with Photoazolol-1 being more active in the dark and Photoazolol-2 demonstrating higher antagonism upon illumination. In addition, we provide a molecular rationale for the interaction of the different photoisomers with the receptor. Overall, we present innovative tools and a proof of concept for the precise control of ß2-AR by means of light.

Research Techniques Made Simple: Deep Learning for the Classification of Dermatological Images.

Deep learning is a branch of artificial intelligence that uses computational networks inspired by the human brain to extract patterns from raw data. Development and application of deep learning methods for image analysis, including classification, segmentation, and restoration, have accelerated in the last decade. These tools have been progressively incorporated into several research fields, opening new avenues in the analysis of biomedical imaging. Recently, the application of deep learning to dermatological images has shown great potential. Deep learning algorithms have shown performance comparable with humans in classifying skin lesion images into different skin cancer categories. The potential relevance of deep learning to the clinical realm created the need for researchers in disciplines other than computer science to understand its fundamentals. In this paper, we introduce the basics of a deep learning architecture for image classification, the convolutional neural network, in a manner accessible to nonexperts. We explain its fundamental operation, the convolution, and describe the metrics for the evaluation of its performance. These concepts are important to interpret and evaluate scientific publications involving these tools. We also present examples of recent applications for dermatology. We further discuss the capabilities and limitations of these artificial intelligence-based methods.

Research Techniques Made Simple: Analysis of Collective Cell Migration Using the Wound Healing Assay.

Collective cell migration is a hallmark of wound repair, cancer invasion and metastasis, immune responses, angiogenesis, and embryonic morphogenesis. Wound healing is a complex cellular and biochemical process necessary to restore structurally damaged tissue. It involves dynamic interactions and crosstalk between various cell types, interaction with extracellular matrix molecules, and regulated production of soluble mediators and cytokines. In cutaneous wound healing, skin cells migrate from the wound edges into the wound to restore skin integrity. Analysis of cell migration in vitro is a useful assay to quantify alterations in cell migratory capacity in response to experimental manipulations. Although several methods exist to study cell migration (such as Boyden chamber assay, barrier assays, and microfluidics-based assays), in this short report we will explain the wound healing assay, also known as the "in vitro scratch assay" as a simple, versatile, and cost-effective method to study collective cell migration and wound healing.

An in vitro priming step increases the expression of numerous epidermal growth and migration mediators in a tissue-engineering construct.

An FDA-approved, prototypic, living, bilayered skin construct (BSC) has been used for non-healing wounds. Using this particular construct as proof of principle, we hypothesized that an in vitro 'priming' step may enhance its repertoire of expression of key mediators and genes. The priming step used here was incubation in Dulbecco's modified Eagle's medium (DMEM) for 24 h at 37°C and 5% CO2 , with or without construct meshing. Microarray and ingenuity pathway analysis (IPA) showed that >1000 genes were overexpressed by the priming step, including interleukin 6 (IL-6), which plays important roles in wound healing. Genes highly overexpressed by priming were those involved in epidermal proliferation and migration. Quantitative real-time PCR (qRT-PCR), immunostaining and western blots verified the results. An epiboly assay (epidermal migration over dermis) showed that BSC epiboly was inhibited by IL-6 neutralizing antibody. Back wounds of nude mice were treated with primed or control BSCs for 3 days prior to harvesting; primed BSCs showed a significantly (p = 0.006) greater level of epidermal migration vs unprimed. Our study demonstrates that an in vitro priming step induces wound healing-related genes in the BSC, leading to a construct that could prove more effective in stimulating wound healing. Copyright © 2014 John Wiley & Sons, Ltd.

Mesenchymal Stem Cells in Chronic Wounds: The Spectrum from Basic to Advanced Therapy.

Significance: Almost 7 million Americans have chronic cutaneous wounds and billions of dollars are spent on their treatment. The number of patients with nonhealing wounds keeps increasing worldwide due to an ever-aging population, increasing number of obese and diabetic patients, and cardiovascular disease. Recent Advances: Advanced treatments for difficult wounds are needed. Therapy with mesenchymal stem cells (MSCs) is attractive due to their differentiating potential, their immunomodulating properties, and their paracrine effects. Critical Issues: New technologies (including growth factors and skin substitutes) are now widely used for stimulating wound healing. However, in spite of these advances, the percentage of complete wound closure in most clinical situations is around 50-60%. Moreover, there is a high rate of wound recurrence. Future Directions: Recently, it has been demonstrated that MSCs speed up wound healing by decreasing inflammation, by promoting angiogenesis, and by decreasing scarring. However, there are some potential limitations to successful MSC therapy. These limitations include the need to improve cell delivery methods, cell viability, heterogeneity in MSC preparations, and suboptimal wound bed preparation. Further large, controlled clinical trials are needed to establish the safety of MSCs before widespread clinical application.

Transforming growth factor beta (TGF-ß) isoforms in wound healing and fibrosis.

Scar formation, with persistent alteration of the normal tissue structure, is an undesirable and significant result of both wound healing and fibrosing disorders. There are few strategies to prevent or to treat scarring. The transforming growth factor beta (TGF-ß) superfamily is an important mediator of tissue repair. Each TGF-ß isoform may exert a different effect on wound healing, which may be context-dependent. In particular, TGF-ß1 may mediate fibrosis in adults' wounds, while TGF-ß3 may promote scarless healing in the fetus and reduced scarring in adults. Thus, TGF-ß3 may offer a scar-reducing therapy for acute and chronic wounds and fibrosing disorders.

Liver enzymes and lipid levels in patients with lipodermatosclerosis and venous ulcers treated with a prototypic anabolic steroid (stanozolol): a prospective, randomized, double-blinded, placebo-controlled trial.

Anabolic steroids have been used to treat lower extremity ulcerations, including venous and cryofibrinogenemic ulcers and lipodermatosclerosis (LDS). Yet there have been no studies to determine the severity and reversibility of side effects of anabolic steroids on liver enzymes and lipid profiles in elderly patients. We therefore evaluated, in a prospective, randomized, double-blinded, placebo-controlled trial, the extent and reversibility of abnormal liver enzymes and lipid profiles in patients with LDS and venous leg ulcers treated with stanozolol at 2 mg twice daily for up to 6 months. Follow-up laboratory testing was done for 2 months after cessation of treatment. A total of 44 patients with LDS and venous ulcers were enrolled and treated with either leg compression alone (placebo) or leg compression plus oral stanozolol 2 mg twice daily (active). Baseline and follow-up laboratory testing of liver enzymes and lipid profiles were obtained. A total of 21 active and 23 placebo patients were treated and evaluated. We measured liver enzymes (aspartate aminotransferase [AST/SGOT], alanine aminotransferase [ALT/SGPT], γ-glutamyl transferase [GGT]) and lipid profile components (high-density lipoprotein [HDL], low-density lipoprotein [LDL], total cholesterol) before, during, and after the treatment period. We found that AST/SGOT and ALT/SGPT became significantly elevated in 29% (P = .0415 at 2 months) and 33% (P = .0182 at 1 month) of patients treated with stanozolol or placebo, respectively, with return to baseline in the posttreatment period. Unexpectedly, 91% of patients on stanozolol developed a significant (P < .0001) decrease in HDL levels, by as much as 37 U/L. All patients remained asymptomatic and levels returned to baseline after discontinuation of the drug. We conclude that low-dose stanozolol, 2 mg twice daily, produces asymptomatic and temporary elevation of liver transaminases and depression of the HDL level in a significant proportion of patients.

Sterol regulatory element binding proteins downregulate LDL receptor-related protein (LRP1) expression and LRP1-mediated aggregated LDL uptake by human macrophages.

OBJECTIVE: In the extracellular intima, extracellular matrix proteoglycans favor LDL retention and aggregation (agLDL). In contrast to native LDL (nLDL), agLDL induces high intracellular cholesteryl ester (CE) accumulation in macrophages. It has been suggested that LDL receptor-related protein (LRP1) is involved in agLDL binding and internalization by macrophages. The aim of this work was to analyze whether sterol regulatory element binding proteins (SREBPs) modulate LRP1 expression and LRP1-mediated agLDL uptake by human monocyte-derived macrophages (HMDM). METHODS AND RESULTS: The treatment of HMDM with small anti-LRP1 interfering RNA (siRNA-LRP1) led to the specific inhibition of LRP1 mRNA expression and also to the inhibition of LRP1 protein expression in these cells. In siRNA-LRP1-treated HMDM, CE accumulation from agLDL uptake (84.66+/-5 microg CE/mg protein) was reduced by 95.76+/-5.22%. This suggests that LRP1 plays a pivotal role in agLDL uptake by HMDM. N-acetyl-leucyl-leucyl-norleucinal (ALLN), an inhibitor of SREBP catabolism, maintained high levels of active SREBP-2 and SREBP-1 even in the presence of nLDL and agLDL. Therefore, ALLN induced LDL receptor (LDLR) upregulation. Concomitantly, a strong downregulation of LRP1 mRNA and LRP1 protein was observed in ALLN-treated macrophages. By decreasing LRP1 expression levels, ALLN reduced CE accumulation from agLDL at all tested concentrations. CONCLUSIONS: These results suggest that high levels of active SREBPs downregulate LRP1 expression and intracellular CE accumulation in HMDM.

Aggregated low density lipoproteins decrease metalloproteinase-9 expression and activity in human coronary smooth muscle cells.

Plaque stability largely depends on vascular smooth muscle cell (VSMC) function. VSMC secrete metalloproteinases (MMPs), matrix degrading endopeptidases, that regulate VSMC migration and function. Among them, gelatinase B or MMP-9 seems to have a protective effect by promoting a stable plaque phenotype. In macrophage foam cells oxidized LDL (oxLDL) uptake regulates MMP-9 expression. However, it is unknown whether VSMC-lipid loading by aggregated LDL (agLDL) internalization produces any effect on MMP-9 production by human resident vascular cells. In the present study, we analyzed the effect of lipid-internalization in MMP-9 and MMP-2 expression and activity and its consequences in VSMC migration. Our results show that agLDL-internalization down-regulates MMP-9 activity in a time-dependent manner up to 42% at 48h and in a dose-dependent manner up to 87% at 300 microg/mL. nLDL induced similar but not sustained decrease on MMP-9 activity. However, neither agLDL nor nLDL exerted any significant effect on MMP-2 and TIMP-1. VSMC regrowth after a scratch injury was significantly reduced by exposure to agLDL. We conclude that agLDL-lipid loading reduces MMP-9 activity and this effect is associated to inhibition of VSMC migration. Thus, agLDL internalization may have consequences on vascular remodeling after injury, and the stability of lipid-rich atherosclerotic plaques.

Cholesteryl esters of aggregated LDL are internalized by selective uptake in human vascular smooth muscle cells.

OBJECTIVE: Low-density lipoprotein (LDL) receptor-related protein (LRP1) mediates the internalization of aggregated LDL (agLDL)-LDL trapped in the arterial intima bound to proteoglycans-into human vascular smooth muscle cells (VSMC). LRP1-mediated agLDL uptake induces high-intracellular cholesteryl ester (CE) accumulation. The aim of this study was to characterize the mechanism of agLDL internalization in human VSMC. METHODS AND RESULTS: The lipidic component of LDL was labeled with [3H] and the apolipoprotein component with [(125)I]. We found that >90% of intracellular CE derived from agLDL uptake was not associated with apoB100 degradation but was selectively taken up from agLDL. The inhibition of LRP1 expression by small interfering RNA treatment led to a decrease of 80+/-0.05% in agLDL-CE selective uptake. AgLDL induced intracellular CE accumulation without a concomitant CE synthesis. Cytosolic and cytoskeletal proteins were not required for CE transport. Electron and confocal microscopy experiments indicate that CE derived from agLDL accumulated in adipophilin-stained lipid droplets that were not removable by high-density lipoprotein. CONCLUSIONS: Taken together, these results demonstrate that LRP1 mediates the selective uptake of CE from agLDL and that CE derived from agLDL is not intracellularly processed but stored in lipid droplets in human VSMC.

Aggregated low-density lipoprotein uptake induces membrane tissue factor procoagulant activity and microparticle release in human vascular smooth muscle cells.

BACKGROUND: Tissue factor (TF) is the main initiator of the arterial blood coagulation system, and aggregated LDL (agLDL) are found in the arterial intima. Our hypothesis is that agLDL internalization by vascular smooth muscle cells (VSMCs) may trigger TF-procoagulant activity. METHODS AND RESULTS: Cultured human VSMCs were obtained from human coronary arteries of explanted hearts during transplant operations. VSMCs were incubated with native LDL (nLDL) or agLDL. TF mRNA was analyzed by real-time polymerase chain reaction, and cellular and released TF protein antigen were analyzed by Western blot. TF microparticle (MP) content was analyzed by flow cytometry and TF activity by a factor Xa generation test. Both nLDL and agLDL strongly and equally increased TF mRNA and cell membrane protein expression, by approximately 5- and 9-fold, respectively. A sustained TF procoagulant activity was induced by agLDL but not by nLDL (agLDL 2.46+/-0.22 versus nLDL 0.72+/-0.12 mU/mg protein at 12 hours). AgLDL increased TF antigen release (agLDL 5.64+/-0.4 versus nLDL 3.28+/-0.22 AU) and TF MP release (agLDL 89.85+/-8.51 versus nLDL 19.69+/-4.59 TF MP/10(3) cells). TF activation and release induced by agLDL is not related to apoptosis. Blockade of LDL receptor-related protein, a receptor for agLDL, prevented the agLDL-induced release of TF protein and TF MP. CONCLUSIONS: VSMC-TF expression is upregulated by both nLDL and agLDL. However, only agLDL engagement to LDL receptor-related protein induced cellular TF procoagulant activity and TF release by human VSMCs.