Association between stroke and venous leg ulcers: A Mendelian randomization study.

To investigate any potential bidirectional causal relationships between stroke and venous leg ulcers (VLUs), Mendelian randomization (MR) analyses were carried out in this study. The exposure factor was stroke, the outcome factor was VLUs. The two-sample MR study was carried out based on the online analysis platform (http://app.mrbase.org/). The association of stroke and VLUs was analysed via methods of Inverse Variance Weighted (IVW), Weighted Median, MR-Egger and weighted mode. IVW method suggested no association between stroke and VLUs ((ß 1.06; SE 9.321; p = 0.9095)). Weighted median estimator (ß 5.906; SE 11.99, p = 0.6223), MR-Egger (ß -0.8677; SE 21.89; p = 0.9691) and weighted mode (ß 9.336; SE 17.77; p = 0.6089) showed consistent results. Conversely, evidence indicating that the presence of VLUs increased the risk of stroke was lacking. According to this MR study, there is no causal connection between stroke and VLUs, which suggests that therapies targeting stroke may not be effective against VLUs.

Association between venous leg ulcers and chronic heart failure: A Mendelian randomization study.

An association between venous leg ulcers (VLU) and chronic heart failure (CHF) has been suggested by observational research. This study used Mendelian randomization (MR) methods to look into any possible bidirectional causal links between VLU and CHF. The 'TwoSampleMR' R package was employed for MR analyses. The association of VLU and CHF was assessed via methods of inverse variance weighted (IVW), weighted mode, MR Egger and weighted median. Results of IVW suggested no association between VLU and CHF (ß 0.008356; SE 0.01889; p = 0.6582). The weighted median estimator (ß -0.005777; SE 0.02059, p = 0.7791), MR-Egger (ß -0.08955; SE 0.04557; p = 0.07296) and weighted mode (ß -0.01202; SE 0.02467; p = 0.6341) showed consistent results. Conversely, evidence indicating that the presence of CHF increased the risk of VLU was lacking. In conclusion, there is no bidirectional causal relationship between VLU and CHF. Further studies are required to validate the findings of this study.

Th17-Gene Expression Profile in Patients with Chronic Venous Disease and Venous Ulcers: Genetic Modulations and Preliminary Clinical Evidence.

Chronic venous disease is a condition globally widespread, resulting in a disabling pathological disorder. The CD4 + Th17+ (Cluster Differentiation 4) lymphocytes represent a regulative factor for innate immunity related to the development of complex diseases. Recently, these mechanisms have been associated with vascular disease. The aim of this work is to validate whether the Th17 response correlates with the development of CVI (Chronic venous insufficiency)and CVLUs (chronic venous limbs ulcers) and whether Th17 markers can be used, both as intrinsic risk factors and diagnostic markers, for disease development. PBL derived from peripheral blood samples of patients and controls were subjected to gene expression analysis for IL23R, IL17, SGK1, TGFß, RORγ, FOXO1, and RANBP1 by qRT-PCR and immunoblot. A post hoc correlation, the diagnostic performance of the target genes, and multivariable analyses were properly conducted. The main expression markers of the CD4 + Th17+ switch were strongly activated in chronic venous insufficiency and in advanced ulceration. The correlation analysis demonstrated the inter-dependence on Th17's signature modulation. ROC (Receiver Operating Characteristic) analysis defined, for the examined genes, a clinical value as the potential diagnostic markers. Multi-logistic regression studies showed that Th17 markers behave as empirical risk factors for CVD (chronic venous disease) development. Taken together, the present data provide a new hypothesis for the TH17-dependent pathogenesis of CVD, favoring the possibility for the development of new diagnostic, preventive, and therapeutic approaches.

microRNA-301a-3p is a potential biomarker in venous ulcers vein and gets involved in endothelial cell dysfunction.

Venous ulcer is a common contributor to chronic venous insufficiency (CVI) of lower limbs, which seriously affects the life quality of patients. In this study, we researched the expression characteristics of microRNA-301a-3p (miR-301a-3p) in patients with CVI and investigated the impact of miR-301a-3p on the dysfunction of human umbilical vein endothelial cells (HUVECs). The plasma level of miR-301a-3p in normal controls, patients with varicose great saphenous vein, and patients with the venous ulcer of lower limbs were measured. We adopted Interleukin-1ß (IL-1ß), H2O2, and oxygen and glucose deprivation (OGD) to induce endothelial cell injury in vitro. In this way, we evaluated the influence of miR-301a-3p on HUVEC viability, apoptosis, inflammatory response, and oxidative stress. Our data showed that miR-301a-3p was substantially overexpressed in patients with lower limb venous ulcers. The viability of HUVECs decreased, and miR-301a-3p was up-regulated after IL-1ß, H2O2, and OGD treatment. miR-301a-3p inhibition greatly ameliorated the dysfunction and cell damage of HUVECs, promoted IGF1/PI3K/Akt/PPARγ, and down-regulated NF-κB/MMPs. The phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002) or the peroxisome proliferator-activated receptor-γ (PPARγ) inhibitor (GW9661) reversed the anti-inflammatory, antioxidant, and anti-apoptotic effects mediated by miR-301a-3p down-regulation. The nuclear factor-κB (NF-κB) inhibitor lessened cell injury mediated by miR-301a-3p overexpression. In terms of the mechanism, miR-301a-3p targeted the 3'UTR of Insulin-like growth factor-1 (IGF1) and repressed the profile of IGF1. Thus, miR-301a-3p mediates venous endothelial cell damage by targeting IGF1 and regulating the IGF1/PI3K/Akt/PPARγ/NF-κB/MMPs pathway.

Periodontal (formerly type VIII) Ehlers-Danlos syndrome: Description of 13 novel cases and expansion of the clinical phenotype.

Periodontal Ehlers-Danlos syndrome (pEDS) is a rare condition caused by pathogenic variants in the C1R and C1S genes, encoding subunits C1r and C1s of the first component of the classical complement pathway. It is characterized by early-onset periodontitis with premature tooth loss, pretibial hyperpigmentation and skin fragility. Rare arterial complications have been reported, but venous insufficiency is rarely described. Here we report 13 novel patients carrying heterozygous pathogenic variants in C1R and C1S including three novel C1S variants (c.962G > C, c.961 T > G and c.961 T > A). In addition to the pEDS phenotype, three patients and one relative displayed widespread venous insufficiency leading to persistent varicose leg ulcers. One patient suffered an intracranial aneurysm with familial vascular complications including thoracic and abdominal aortic aneurysm and dissection and intracranial aneurysm rupture. This work confirms that vascular complications can occur, although they are not frequent, which leads us to propose to carry out a first complete non-invasive vascular evaluation at the time of the diagnosis in pEDS patients. However, larger case series are needed to improve our understanding of the link between complement pathway activation and connective tissue alterations observed in these patients, and to better assess the frequency, type and consequences of the vascular complications.

A possible role for inducible arginase isoform (AI) in the pathogenesis of chronic venous leg ulcer.

Chronic venous ulcer (CVU) is a major cause of chronic wounds of lower extremities and presents a significant financial and resource burden to health care systems worldwide. Defects in the vasculature, matrix deposition, and re-epithelialization are the main histopathological changes believed to impede healing. Supplementation of the amino acid arginine that plays a crucial role in the interactions that occur during inflammation and wound healing was proven clinically to improve acute wound healing probably through enhancing activity of inducible arginase (AI) locally in the wounds. However, the possible mechanism of arginine action and the potential beneficial effects of AI/arginine in human chronic wounds remain unclear. In the present study, using biopsies, taken under local anesthesia, from adult patients (n = 12, mean age 55 years old) with CVUs in lower extremities, we investigated the correlation between AI distribution in CVUs and the histopathological changes, mainly proliferative and vascular changes. Our results show a distinct spatial distribution of AI along the ulcer in the epidermis and in the dermis with the highest level of expression being at the ulcer edge and the least expression towards the ulcer base. The AI cellular immunoreactivity, enzymatic activity, and protein levels were significantly increased towards the ulcer edge. Interestingly, a similar pattern of expression was encountered in the proliferative and the vascular changes with strong correlations between AI and the proliferative activity and vascular changes. Furthermore, AI cellular distribution was associated with increased proliferative activity, inflammation, and vascular changes. Our findings of differential expression of AI along the CVU base, edge, and nearby surrounding skin and its associations with increased proliferative activity and vascular changes provide further support to the AI implication in CVU pathogenesis. The presence of high levels of AI in the epidermis of chronic wounds may serve as a molecular marker of impaired healing and may provide future targets for therapeutic intervention.

A bioengineered living cell construct activates metallothionein/zinc/MMP8 and inhibits TGFß to stimulate remodeling of fibrotic venous leg ulcers.

Venous leg ulcers (VLU) represent a major clinical unmet need, impairing quality of life for millions worldwide. The bioengineered bilayered living cell construct (BLCC) is the only FDA-approved therapy demonstrating efficacy in healing chronic VLU, yet its in vivo mechanisms of action are not well understood. Previously, we reported a BLCC-mediated acute wounding response at the ulcer edge; in this study we elucidated the BLCC-specific effects on the epidermis-free ulcer bed. We conducted a randomized controlled clinical trial (ClinicalTrials.gov NCT01327937) enrolling 30 subjects with nonhealing VLUs, and performed genotyping, genomic profiling, and functional analysis on wound bed biopsies obtained at baseline and 1 week after treatment with BLCC plus compression or compression therapy (control). The VLU bed transcriptome featured processes of chronic inflammation and was strikingly enriched for fibrotic/fibrogenic pathways and gene networks. BLCC application decreased expression of profibrotic TGFß1 gene targets and increased levels of TGFß inhibitor decorin. Surprisingly, BLCC upregulated metallothioneins and fibroblast-derived MMP8 collagenase, and promoted endogenous release of MMP-activating zinc to stimulate antifibrotic remodeling, a novel mechanism of cutaneous wound healing. By activating a remodeling program in the quiescent VLU bed, BLCC application shifts nonhealing to healing phenotype. As VLU bed fibrosis correlates with poor clinical healing, findings from this study identify the chronic VLU as a fibrotic skin disease and are first to support the development and application of antifibrotic therapies as a successful treatment approach.

Genetic biases related to chronic venous ulceration.

Chronic wounds represent a major socioeconomic problem. Chronic venous ulceration is one of the least well-understood types of chronic wounds. A chronic venous ulcer arises as a result of chronic venous insufficiency (CVI), which affects approximately 10-35% of people in the developed world, yet not all people with CVI develop ulceration. The question of why some patients with CVI develop chronic ulceration and others do not, still remains unanswered. Risk factors for the development of chronic ulceration are poorly understood and include age, residual iliofemoral vein obstruction, residual deep incompetence, persistent venous hypertension, obesity and genetics. The genetic aspects of CVI have only been vaguely evaluated. This paper reports on a literature review of the variation in genetic polymorphisms and gene expression associated with the development of a chronic venous ulceration.

WAKMAR2, a Long Noncoding RNA Downregulated in Human Chronic Wounds, Modulates Keratinocyte Motility and Production of Inflammatory Chemokines.

Chronic wounds represent a major and growing health and economic burden worldwide. A better understanding of molecular mechanisms of normal as well as impaired wound healing is needed to develop effective treatment. Herein we studied the potential role of long noncoding RNA LOC100130476 in skin wound repair. LOC100130476 is an RNA polymerase II-encoded polyadenylated transcript present in both cytoplasm and nucleus. We found that its expression was lower in wound-edge keratinocytes of human chronic wounds compared to normal wounds of healthy donors and intact skin. In cultured keratinocytes, LOC100130476 expression was induced by TGF-ß signaling. By reducing LOC100130476 expression with antisense oligos or activating its transcription with CRISPR/Cas9 Synergistic Activation Mediator system, we showed that LOC100130476 restricted the production of inflammatory chemokines by keratinocytes, while enhancing cell migration. In line with this, knockdown of LOC100130476 impaired re-epithelization of human ex vivo wounds. Based on these results, we named LOC100130476 wound and keratinocyte migration-associated long noncoding RNA 2 (WAKMAR2). Moreover, we identified a molecular network that may mediate the biological function of WAKMAR2 in keratinocytes using microarray. In summary, our data suggest that WAKMAR2 is an important regulator of skin wound healing and its deficiency may contribute to the pathogenesis of chronic wounds.

A systematic review on risk factors in developing a first-time Venous Leg Ulcer.

Venous Leg Ulcers (VLU) occur in about 1% of the Western population. A VLU takes 3-12 months to heal, it recurs often, and it has a negative impact on the quality of life. The risk factors for the development of a first VLU are not well-understood and prevention of a first VLU therefore remains underappreciated. The aim of this study was to identify risk factors for developing a first VLU in adults (aged > 18 years) by searching the literature. We searched the Cochrane Library, Pubmed, Cinalh and Narcisto identify studies that investigated risk factors in developing a VLU. The last search was performed in January 2018. Two reviewers independently reviewed the abstracts and full-text articles, and assessed the methodological quality of the included studies. Results of studies using duplex scanning, and comparing participants with and without VLUs were included in the qualitative analysis. Where possible a quantitative meta-analysis was conducted. We found five studies that investigated the relation of several risk factors with VLU development. The methodological differences of the studies made it impossible to perform a quantitative analysis. The risk factors higher age (four studies), higher body mass index (four studies), low physical activity (four studies), arterial hypertension (four studies), deep vein reflux (three studies), deep venous thrombosis (three studies) and family history of VLU (three studies) were significantly associated with a VLU in the majority of the studies. To what extent they influence the development of a VLU remains unclear because of the limited number of studies that investigated the association of these risk factors with VLU development, and the heterogeneity of these studies. Further studies are needed to confirm the association of these risk factors with the development of a VLU and to explore overweight and low physical activity in more detail.

Expression of the SOCS family in human chronic wound tissues: Potential implications for SOCS in chronic wound healing.

Cytokines play important roles in the wound healing process through various signalling pathways. The JAK-STAT pathway is utilised by most cytokines for signal transduction and is regulated by a variety of molecules, including suppressor of cytokine signalling (SOCS) proteins. SOCS are associated with inflammatory diseases and have an impact on cytokines, growth factors and key cell types involved in the wound­healing process. SOCS, a negative regulator of cytokine signalling, may hold the potential to regulate cytokine­induced signalling in the chronic wound­healing process. Wound edge tissues were collected from chronic venous leg ulcer patients and classified as non-healing and healing wounds. The expression pattern of seven SOCSs members, at the transcript and protein level, were examined in these tissues using qPCR and immunohistochemistry. Significantly higher levels of SOCS3 (P=0.0284) and SOCS4 (P=0.0376) in non-healing chronic wounds compared to the healing/healed chronic wounds were observed at the transcript level. Relocalisation of SOCS3 protein in the non-healing wound environment was evident in the investigated chronic biopsies. Thus, the results show that the expression of SOCS transcript indicated that SOCS members may act as a prognostic biomarker of chronic wounds.

Use of 16S rRNA sequencing and quantitative PCR to correlate venous leg ulcer bacterial bioburden dynamics with wound expansion, antibiotic therapy, and healing.

Clinical diagnosis of infection in chronic wounds is currently limited to subjective clinical signs and culture-based methods that underestimate the complexity of wound microbial bioburden as revealed by DNA-based microbial identification methods. Here, we use 16S rRNA next generation sequencing and quantitative polymerase chain reaction to characterize weekly changes in bacterial load, community structure, and diversity associated with a chronic venous leg ulcer over the 15-week course of treatment and healing. Our DNA-based methods and detailed sampling scheme reveal that the bacterial bioburden of the wound is unexpectedly dynamic, including changes in the bacterial load and community structure that correlate with wound expansion, antibiotic therapy, and healing. We demonstrate that these multidimensional changes in bacterial bioburden can be summarized using swabs taken prior to debridement, and therefore, can be more easily collected serially than debridement or biopsy samples. Overall, this case illustrates the importance of detailed clinical indicators and longitudinal sampling to determine the pathogenic significance of chronic wound microbial dynamics and guide best use of antimicrobials for improvement of healing outcomes.

Genetic polymorphisms of vein wall remodeling in chronic venous disease: a narrative and systematic review.

Chronic venous disease encompasses a spectrum of disorders caused by an abnormal venous system. They include chronic venous insufficiency, varicose veins, lipodermatosclerosis, postthrombotic syndrome, and venous ulceration. Some evidence suggests a genetic predisposition to chronic venous disease from gene polymorphisms associated mainly with vein wall remodeling. The literature exploring these polymorphisms has not been reviewed and compiled thus far. In this narrative and systematic review, we present the current evidence available on the role of polymorphisms in genes involved in vein wall remodeling and other pathways as contributors to chronic venous disease. We searched the EMBASE, Medline, and PubMed databases from inception to 2013 for basic science or clinical studies relating to genetic associations in chronic venous disease and obtained 38 relevant studies for this review. Important candidate genes/proteins include the matrix metalloproteinases (extracellular matrix degradation), vascular endothelial growth factors (angiogenesis and vessel wall integrity), FOXC2 (vascular development), hemochromatosis (involved in venous ulceration and iron absorption), and various types of collagen (contributors to vein wall strength). The data on associations between these genes/proteins and the postthrombotic syndrome are limited and additional studies are required. These associations might have future prognostic and therapeutic implications.

Omics profiles in chronic venous ulcer wound fluid: innovative applications for translational medicine.

Chronic venous disease represents a healthcare problem due to high prevalence and recurrence rates. Studies on chronic venous ulcer wound fluid (CVUWF) have demonstrated increased inflammation and proteolysis which can cause tissue destruction and delayed healing. This review discusses: nearly all known metabolites discovered in the past 25 years in CVUWF studies; the omics approaches characterizing the microenvironment of human venous leg ulcers; and the use of biocompounds as prognostic biomarkers and as possible targets for therapeutic approaches. A biomarker is a biological compound that can be functional or non-functional, specific or non-specific in the diagnosis/prognosis to a disease state and may be quantified to determine progression or regression of disease. Omics studies in CVUWF provide the impetus for future identification of biomarkers within the intricate network in chronic venous disease and set the basis for determining the appropriate combination of molecules that are expressed with the healing status of venous leg ulcers.

Targeting Cx43 and N-cadherin, which are abnormally upregulated in venous leg ulcers, influences migration, adhesion and activation of Rho GTPases.

BACKGROUND: Venous leg ulcers can be very hard to heal and represent a significant medical need with no effective therapeutic treatment currently available. PRINCIPAL FINDINGS: In wound edge biopsies from human venous leg ulcers we found a striking upregulation of dermal N-cadherin, Zonula Occludens-1 and the gap junction protein Connexin43 (Cx43) compared to intact skin, and in stark contrast to the down-regulation of Cx43 expression seen in acute, healing wounds. We targeted the expression of these proteins in 3T3 fibroblasts to evaluate their role in venous leg ulcers healing. Knockdown of Cx43 and N-cadherin, but not Zonula Occludens-1, accelerated cell migration in a scratch wound-healing assay. Reducing Cx43 increased Golgi reorientation, whilst decreasing cell adhesion and proliferation. Furthermore, Connexin43 and N-cadherin knockdown led to profound effects on fibroblast cytoskeletal dynamics after scratch-wounding. The cells exhibited longer lamelipodial protrusions lacking the F-actin belt seen at the leading edge in wounded control cells. This phenotype was accompanied by augmented activation of Rac-1 and RhoA GTPases, as revealed by Förster Resonance Energy Transfer and pull down experiments. CONCLUSIONS: Cx43 and N-cadherin are potential therapeutic targets in the promotion of healing of venous leg ulcers, by acting at least in part through distinct contributions of cell adhesion, migration, proliferation and cytoskeletal dynamics.

Prevalence rate for inherited thrombophilia in patients with chronic and recurrent venous leg ulceration.

The aim of the study was to determine the prevalence rate for inherited thrombophilia (IT) in patients with chronic (CVU) and recurrent venous leg ulceration. We also investigated and evaluated the severity of the clinical pattern of CVU in patients with and without IT. We examined 110 patients with CVU (the study group) and 110 healthy subjects (the control group). We prepared a questionnaire to be completed by each study participant. Ultrasound Doppler color imaging or/and duplex ultrasonography was performed to evaluate the efficiency of the venous system. The ankle-brachial index was calculated to determine the efficiency of the arterial system. We examined both groups for the presence of IT. IT was diagnosed in 30% of study group and in 1.8% of control group. Our diagnoses of deep vein thrombosis (DVT) were based on medical interviews, physical examinations, and an ultrasonography of the venous system and concerned 64 study group patients (58.2%), 35 of whom (31.8%) experienced recurrent DVT. Proximal and/or distal DVT was determined in an interview and/or by an ultrasonography performed for all patients with CVU and IT. In 94% of these patients, DVT was recurrent, and in 88% of patients with CVU and IT, we observed recurrent DVT and CVU. It recurred more often and persisted longer when compared to patients with CVU and no IT, despite similar management. No differences were observed in ulcer size, localization, or pain level related to ulceration between patients with CVU and IT and those with CVU and no IT.

Activated leukocyte cell adhesion molecule impacts on clinical wound healing and inhibits HaCaT migration.

Activated leukocyte cell adhesion molecule (ALCAM) is a glycoprotein of the immunoglobulin superfamily that has been implicated in the processes of cell adhesion and migration. The current study examines the importance of ALCAM in regulating HaCaT cell growth and migration and its potential to impact on wound healing. ALCAM levels were examined in a range of clinical wound and normal skin samples using Q-PCR and immunohistochemistry. ALCAM expression was targeted in HaCaT keratinocyte cells using a hammerhead ribozyme transgene system. Subsequently, the impact of ALCAM suppression on HaCaT migration and growth was assessed. ALCAM protein was detected mainly in keratinocytes. ALCAM transcript levels were found to be significantly higher in the non-healed chronic wound samples compared with healed samples (P = 0·026). In addition, targeting of ALCAM in HaCaT cells brought about a substantial increase in cellular migration and growth compared with HaCaT control cells.Our results suggest that ALCAM plays an important role in the migration of HaCaT keratinocyte cells. The data also suggests that higher levels of ALCAM may impair healing in chronic wounds. The impact of ALCAM in wound healing may thus be somewhat due to its impact on cell migration and growth.

Interplay of iron metallobiology, metalloproteinases, and FXIII, and role of their gene variants in venous leg ulcer.

The iron metallobiology has long been suspected as a causal agent in venous leg ulcer (VLU) pathophysiology. However, it was demonstrated only recently that visible iron deposits cause lesions in only some individuals due to functional iron and related gene variants. In this article, the mechanism by which dysregulated iron cycle leads to local iron overload that could generate free radicals or activate a proteolytic hyperactivity on the part of matrix metalloproteinases (MMPs) or else downregulate tissue inhibitors of MMPs is reviewed. Also reviewed is the interplay of other vital factors such as coagulation factor XIII (FXIII), which influences tissue remodeling and angiogenesis, leading to impaired healing of the lesion, whether there exists altered interaction with MMPs or in presence of particular unfavorable single nucleotide polymorphisms.