Oxidized Low-Density Lipoprotein Induces WNT5A Signaling Activation in THP-1 Derived Macrophages and a Human Aortic Vascular Smooth Muscle Cell Line.

The pathogenesis of atherosclerosis is complex, evolves, and involves many cell types. Macrophages and vascular smooth muscle cells (VSMCs) are critically involved in atherosclerosis development and progression. Several studies have shown that WNT5A protein is abundantly expressed in human atherosclerotic lesions; however, the mechanism and role of WNT signaling pathway activation is not clearly known. Using THP-1 derived macrophages, and human aortic VSMC cells, we evaluated in vitro how oxidized low-density lipoprotein (oxLDL) and WNT5A signaling interact in these two cell lines. We used western blot, scratch assay, metabolic proliferation assay, as well as immunostaining to analyze the effect of Wnt signaling activation. The results demonstrated that oxLDL, as well as WNT5A (control), induced Disheveled-2 (DVL2) activation and Kif26b degradation, indicating activation of non-canonical Wnt signaling. We found that oxLDL and WNT5A induced FZD5-ROR2 co-localization at the cellular membrane in vitro in THP-1 derived macrophages. Box5 (FZD5 receptor antagonist) inhibited oxLDL-induced DVL2/JNK activation secondary to newly secreted WNT protein from THP-1 derived macrophages. We found that WNT3A (canonical Wnt) and WNT5A showed different roles in this VSMC cell line. These findings indicate that WNT5A is upregulated by oxLDL, promotes foam cell formation, and affects VSMC phenotype and migration in these two cell lines. Also, in these cell lines FZD5 signaling seems to be necessary for lipid accumulation and, through this mechanism, WNT5A could modulate foam cell formation. Thus, our results suggest that WNT5A may contribute to the pathogenesis of vascular disease through modulating macrophage and VSMC behavior.

Targeting Dysfunctional Vascular Endothelial Cells Using Immunoliposomes Under Flow Conditions.

INTRODUCTION: Atherosclerosis (ATH), the build up of fat in the arteries, is a principal cause of heart attack and stroke. Drug instability and lack of target specificity are major drawbacks of current clinical therapeutics. These undesirable effects can be eliminated by site-specific drug delivery. The endothelial surface over ATH lesions has been shown to overexpress vascular cell adhesion molecule1 (VCAM1), which can be used for targeted therapy. METHODS: Here, we report the synthesis, characterization, and development of anti VCAM1-functionalized liposomes to target cells overexpressing VCAM1 under static and flow conditions. Liposomes were composed of dioleoyl-phosphatidylcholine, sphingomyelin, cholesterol, and distearoyl-phosphatidylethanolamine-polyethylene glycol-cyanur (31.67:31.67:31.67:5 mol%). VCAM1 expression in endothelial cells was induced by lipopolysaccharide (LPS) treatment. RESULTS: Characterization study revealed that liposomes were negatively charged (- 7.7 ± 2.6 mV) with an average diameter of 201.3 ± 3.3 nm. Liposomes showed no toxicity toward THP-1 derived macrophages and endothelial cells. Liposomes were able to target both fixed and non-fixed endothelial cells, in vitro, with significantly higher localization observed in non-fixed conditions. To mimic biological and physiologically-relevant conditions, liposome targeting was also examined under flow (4 dyn/cm2) with or without erythrocytes (40% v/v hematocrit). Liposomes were able to target LPS-treated endothelial cells under dynamic culture, in the presence or absence of erythrocytes, although targeting efficiency was five-fold lower in flow compared to static conditions. CONCLUSIONS: This liposomal delivery system showed a significant improvement in localization on dysfunctional endothelium after surface functionalization. We conclude that VCAM1-functionalized liposomes can target and potentially deliver therapeutic compounds to ATH regions.

Blocking Wnt5a signaling decreases CD36 expression and foam cell formation in atherosclerosis.

BACKGROUND AND AIMS: Wnt5a is a highly studied member of the Wnt family and recently has been implicated in the pathogenesis of atherosclerosis, but its precise role is unknown. Foam cell development is a critical process to atherosclerotic plaque formation. In the present study, we investigated the role of noncanonical Wnt5a signaling in the development of foam cells. METHODS: Human carotid atherosclerotic tissue and THP-1-derived macrophages were used to investigate the contribution of Wnt5a signaling in the formation of foam cells. Immunohistochemistry was used to evaluate protein expression of scavenger receptors and noncanonical Wnt5a receptors [frizzled 5 (Fz5) and receptor tyrosine kinase-like orphan receptor 2 (Ror2)] in human atherosclerotic macrophages/foam cells. Changes in protein expression in response to Wnt5a stimulation/inhibition were determined by Western blot, and lipid accumulation was evaluated by fluorescent lipid droplet staining. RESULTS: Wnt5a (P<.05), Fz5 (P<.01), and Ror2 (P<.01) were significantly expressed in advanced atherosclerotic lesions compared to less advanced lesions (N=10). Wnt5a, Fz5, and Ror2 were expressed in macrophages/foam cells within the plaque. In vitro studies revealed that Wnt5a significantly increased the expression of the lipid uptake receptor CD36 (P<.05) but not the lipid efflux receptor ATP-binding cassette transporter (P>.05). rWnt5a also significantly increased lipid accumulation in THP-1 macrophages (P<.05). Furthermore, inhibition of Wnt5a signaling with Box5 prevented lipid accumulation (P<.01) and prevented CD36 up-regulation (P<.01). CONCLUSIONS: These results suggest a direct role for Wnt5a signaling in the pathogenesis of atherosclerosis, specifically the accumulation of lipid in macrophages and the formation of foam cells.

Interrelationship of canonical and non-canonical Wnt signalling pathways in chronic metabolic diseases.

Chronic diseases account for approximately 45% of all deaths in developed countries and are particularly prevalent in countries with the most sophisticated and robust public health systems. Chronic metabolic diseases, specifically lifestyle-related diseases pertaining to diet and exercise, continue to be difficult to treat clinically. The most prevalent of these chronic metabolic diseases include obesity, diabetes, non-alcoholic fatty liver disease, chronic kidney disease and cardiovascular disease and will be the focus of this review. Wnt proteins are highly conserved glycoproteins best known for their role in development and homeostasis of tissues. Given the importance of Wnt signalling in homeostasis, aberrant Wnt signalling likely regulates metabolic processes and may contribute to the development of chronic metabolic diseases. Expression of Wnt proteins and dysfunctional Wnt signalling has been reported in multiple chronic diseases. It is interesting to speculate about an interrelationship between the Wnt signalling pathways as a potential pathological mechanism in chronic metabolic diseases. The aim of this review is to summarize reported findings on the contrasting roles of Wnt signalling in lifestyle-related chronic metabolic diseases; specifically, the contribution of Wnt signalling to lipid accumulation, fibrosis and chronic low-grade inflammation.

Wnt5a / planar cell polarity signaling pathway in urothelial carcinoma, a potential prognostic biomarker.

Bladder cancer is the fourth most common cancer in men and the most common malignancy of the urinary tract. Bladder cancers detected at an early stage have a very high five-year survival rate, but when detected after local metastasis the rate is only about 50%. Our group recently reported a positive correlation between the expression of Wnt5a, a member of the Wnt proteins family, and histopathological grade and stage of urothelial carcinoma (UC). The objective of this study was to analyze UC cases reported in Athens, Ohio and investigate the major components of Wnt5a / planar cell polarity (PCP) signaling pathway in UC human tissue samples and UC cell lines.Formalin fixed and paraffin embedded transurethral resection tissues were immunostained for Wnt5a, Ror-2, CTHRC1 and E-cadherin. In addition, in vitro studies using UC cell lines were investigated for Wnt5a/PCP signaling and epithelial mesenchymal transition (EMT) gene expression. The IHC results showed a correlation between the expression of Wnt5a, Ror2 and CTHRC1 with high histological grade of the tumor, while E-cadherin showed an opposite trend of expression. Real time RT-PCR results showed that RNA expression of the Wnt5a/ PCP pathway genes vary in low and high grade UC cell lines and that the high grade cell lines exhibited signs of EMT.These findings support that Wnt5a-Ror2 signaling plays a role in UC, support the potential use of Wnt5a as a prognostic marker and provide evidence that Wnt5a signaling may be used as an effective molecular target for novel therapeutic tools.

Effectiveness of Home Blood Pressure Monitoring Among Low-Income Adults in Rural Appalachia.

CONTEXT: High blood pressure (BP) is a common chronic condition in the United States. For many people, BP control through pharmacologic intervention alone is not effective at maintaining a healthy BP. Team-based, patient-focused care and home-based BP monitoring in addition to pharmacologic interventions have been shown to be effective for controlling BP. OBJECTIVE: To determine the effectiveness of the hypertension management program at the Heritage Community Clinic in Athens, Ohio. METHODS: Medical records of 43 patients who took part in the hypertension management program were retrospectively reviewed and included clinical data such as age, sex, BP, body mass index, comorbidities, family history, and demographic information. In addition to standard pharmacologic interventions, the program provided equipment for at-home BP monitoring, education on behavior and lifestyle modification, and 5 follow-up visits. Data from the 5 follow-up visits were analyzed. RESULTS: Linear mixed-effects regression models of BP suggested that the visit factor was significantly associated with BP (P<.001). On average at each visit, patients showed a 6.8-mm Hg reduction in systolic BP and a 3.8-mm Hg reduction in diastolic BP after controlling for demographic variables. General stress level, marital status, and depression were all significantly associated with BP (P<.05). In addition, 67.5% of the patients who took part in this program achieved the target treatment guidelines of the Eighth Joint National Committee for hypertension management. CONCLUSION: A clinic-based hypertension management program comprising patient education, support, medication, and home BP monitoring was effective at reducing BP.