Performing Department
Nutrition and Food Science
Non Technical Summary
Cardiovascular disease is the number one cause of death in the U.S. Atherosclerosis, a chronic inflammatory disease of the aorta, is responsible for most of the mortality and morbidity associated with cardiovascular disease. Activation of macrophages and smooth muscle cells (SMCs), and formation of lipid-laden foam cells, are linked to atherosclerosis. Recent studies also suggest that arterial stiffness is a risk factor for atherosclerosis. We developed ApoE:TRPV4 double null mouse models for directly testing the role of TRPV4 (transient receptor potential vanilloid 4), a Ca2+-permeable ion channel, and a known matrix stiffness sensor, in high-fat diet-induced atherosclerosis. Based on our published and preliminary data, our central hypothesis is that SMC-TRPV4 mediates inflammatory and atherogenic activity in response to hyperlipidemia, and this activity promotes atherosclerosis development and progression in a manner dependent on matrix stiffness. We will utilize innovative molecular and genetic tools, and in vivo and in vitro models to test the hypothesis. We expect that the results of this study will provide novel information and insight regarding the mechanisms of regulation of high-fat diet-induced atherogenesis, and will potentially identify a targetable receptor/pathway for the amelioration of atherosclerosis.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The overall goal of our research is to uncover the molecular mechanisms underlying atherosclerosis and to identify potential therapeutic targets. To investigate this, we have developed a novel ApoE:TRPV4 double-knockout mouse models to directly assess the role of TRPV4--a Ca2+-permeable ion channel and known matrix stiffness sensor--in high-fat diet-induced atherosclerosis. Preliminary findings indicate a connection between TRPV4, aortic matrix stiffness, and the development of atherosclerosis. Based on published data and our preliminary results, we hypothesize that SMC-TRPV4 mediates inflammatory and atherogenic activity in response to hyperlipidemia, and this activity promotes atherosclerosis development and progression in a manner dependent on matrix stiffness. We will use advanced molecular and genetic techniques, along with in vivo and in vitro models, to test this hypothesis through the following two research objectives.Objective 1: Generation of TRPV4flox/floxSM22-Cre (TRPV4SMC/null), ApoEnull:TRPV4SMC/null, and ApoEnull:TRPV4flox/flox (control) mouse.Objective 2: Determine the effect of SMC-TRPV4 deficiency on macrophage and SMC accumulation, inflammation, foam cell formation, aortic stiffening, and aortic plaque development using ApoEnull:TRPV4SMC/null and ApoEnull:TRPV4flox/flox (control) mouse models.
Project Methods
Experimental Design-1: Since TRPV4 is also expressed in SMC, to determine if the effect of TRPV4 on atherosclerosis in vivo is specifically attributable to SMC-TRPV4, our TRPV4flox/flox mice will be crossed with SM22-Cre mice (Cat# 017491, The Jackson Lab) to create an SMC-specific TRPV4 knockout (TRPV4SMC/null) mouse. Here's a brief step-by-step protocol: 1) We will cross TRPV4flox/flox mice with SM22-Cre mice. These cross aims to produce offspring that carry both the floxed TRPV4 allele and the SM22-Cre transgene. 2) At weaning age, we will collect tail snips from the F1 offspring for genotyping. 3) We will conduct PCR analysis to identify heterozygous F1 (TRPV4flox/+:SM22-Cre) mice that have inherited one copy of the floxed TRPV4 allele and the Cre recombinase gene. 4) We will breed these TRPV4flox/+:SM22- Cre mice together to obtain some offspring that are homozygous for the floxed allele (TRPV4flox/flox) and carry the SM22-Cre transgene. 5) We will collect tissue samples from F2 offspring for genotyping at weaning. 6) We will perform PCR to identify mice homozygous for the floxed TRPV4 allele (TRPV4flox/flox) that also carry the SM22-Cre gene. These mice are the desired TRPV4SMC/null mice, with a homozygous floxed TRPV4 allele specifically knocked out in SMCs due to the presence of Cre recombinase. 7) To confirm the SMC-specific knockout of TRPV4, we will perform PCR or immunohistochemistry on smooth muscle tissues from the generated mice. These analyses should demonstrate the absence of TRPV4 expression specifically in SMCs. We will confirm the deficiency of TRPV4 function (Ca2+ influx) in SMC harvested from TRPV4SMC/null mice compared to TRPV4flox/flox mice. These mice will be crossed to ApoE null mice, a standard mouse model to study atherosclerosis, to generate ApoEnull:TRPV4SMC/null, and ApoEnull:TRPV4flox/flox (control) mouse. We expect to generate ApoEnull:TRPV4SMC/null mouse in which we will be able to knockout TRPV4 expression specifically in SMCs. We have extensive experience, and expertise in generating cell-specific knockout mouse, thus, we do not anticipate any difficulties performing the experiments proposed in this Aim.Experimental Design-2: For this, randomly selected, age and sex-matched ApoEnull:TRPV4flox/flox (control) and ApoEnull:TRPV4SMC/null mice (6 week old mice, n = 24 mice/group, 12 males and 12 females; 6 mice/time point) will be maintained on a normal chow diet (control) or high fat diet (Harlan Teklad, Madison, WI). Six different multidisciplinary measurements for readouts of atherosclerotic and metabolic changes will be done by collecting various samples from experimental and control mice:Examine the extent/morphology of atherosclerotic lesions (on weeks 6 and 12): For aortic tree analysis, the entire aorta from the heart to 5-10 mm below the bifurcation of the iliac arteries will be removed, dissected, opened longitudinally, and evaluated for lesion area by en face Oil-Red-O staining. For aortic root analysis, serial sections will be taken at the level of the aortic leaflet and stained with Oil-Red-O. We will also examine the abundance of SMCs (by a-SMA), macrophages (by CD68 or Mac3), total collagen (by hydroxyproline), and apoptosis (by TUNEL assay) in aortic root sections/tissues as we have published.Serum lipid profile: We will examine plasma lipid profiles at the beginning of the diet and immediately prior to sacrifice. Cholesterol and triacylglycerol levels will be determined using enzymatic kits (Sigma), and lipoprotein analysis will be carried out by Liquid Chromatography as we have published.Analysis of metabolic parameters: We will examine i) energy intake (kj/day), ii) insulin and glucose levels in blood under fasting and fed condition, ii) subcutaneous, visceral, and liver fat content, and iii) plasma levels of non-esterified fatty acids.Inflammation: Readouts for inflammation on weeks 0, 6, and 12 will be total cell counts and differentials (macrophages and lymphocytes), and total protein (TNFα, IL6, IL1β, IL12, and MCP-1) in the aortic tissues and plasma using our published methods.Aortic tissue stiffening analysis by atomic force microscopy (AFM): For this, 30 μm aortic arch, roots, and descending aorta sections will be subjected to AFM analysis. Briefly, for AFM analysis, we will use the JPK Nano Wizard 4 atomic force microscope from Bruker Nano GmbH in Berlin, Germany (available in our Fischell Dept. of Bioengineering, UMCP), to measure the stiffness of aortic tissues. For this purpose, we will utilize 30 μm-thick aorta sections immobilized on glass slides submerged in PBS to record individual force spectroscopy curves (F-D curves) in contact mode for each sample. To perform these measurements, we will use CP-qp-CONT- BSG-B-5 colloidal probes (sQube), which had specific characteristics, including a nominal resonance frequency of 30 kHz in air, a spring constant of 0.1 N/m, gold coating on the detector side, and an attached borosilicate glass sphere with a diameter of 10 μm. We will set a relative set point of 1 nN, an extension speed of 10 μm/s, and a Z length of 10 μm to ensure appropriate F-D curves. A total of 500 F-D curves will be recorded across various areas surrounding each tissue sample (n = 5 mice per sample). Subsequently, these curves will be processed using the Hertz contact mechanics model for spherical probes to determine the Young's modulus (E). For data visualization and analysis, we will use Graph Pad Prism to generate histograms, bar plots, and line plots for each condition.Foam cell formation: By analysis of Oil-Red-O+ macrophages, as we have published previously.Based on our preliminary studies, we anticipate that macrophage and SMC accumulation, inflammatory protein expression, foam cell formation, aortic stiffening, and atherosclerotic lesion formation in response to hyperlipidemia will be dependent on the presence of TRPV4 in SMCs. Given that TRPV4 is a mechanically sensitive channel, we also predict that in the absence of TRPV4 in SMCs, aortic stiffness-induced macrophage and SMC accumulation will be attenuated. We have extensive experience, and expertise, in atherosclerosis studies using in vitro and in vivo models, thus, we do not anticipate any difficulties performing the experiments proposed in this Aim.