WHAT A DIFFERENCE HYPERTENSION MAKES: THE ADVENTITIA HANDS OFF TO PVAT FOR EXTRACELLULAR MATRIX REMODELING AS BLOOD PRESSURE RISES IN THE HIGH FAT FED DAHL SS RAT By Caitlin Wilson A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Laboratory Research in Pharmacology and Toxicology – Master of Science 2023 ABSTRACT Tunica media extracellular matrix (ECM) remodeling is well understood to occur in response to elevated blood pressure, unlike the remodeling of other tunicae. We hypothesize that perivascular adipose tissue (PVAT) is responsive to hypertension and remodels as a protective measure. The adventitia and PVAT of the thoracic aorta were used in measuring ECM genes from 5 pairs of Dahl SS male rats on 8 or 24 weeks of feeding from weaning on a control (10% Kcal fat) or high fat (HF; 60%) diet. A PCR array of ECM genes was performed with cDNA from adventitia and PVAT after 8 and 24 weeks. A gene regulatory network of the differentially expressed genes (DEGs) (HF 2-fold > Con) was created using Cytoscape. After 8 weeks, 29 adventitia but 0 PVAT DEGs were found. By contrast, at 24 weeks, PVAT possessed 47 DEGs while adventitia had 3. Top DEGs at 8 weeks in adventitia were thrombospondin 1 and collagen 8a1. At 24 weeks, thrombospondin 1 was also a top DEG in PVAT. The transcription factor Adarb1 was identified as a regulator of DEGs in 8 week adventitia and 24 week PVAT. These data support that PVAT responds biologically once blood pressure is elevated. TABLE OF CONTENTS INTRODUCTION .......................................................................................................................... 1 MATERIALS AND METHODS .................................................................................................... 3 RESULTS ....................................................................................................................................... 8 DISCUSSION ............................................................................................................................... 10 CONCLUSIONS .......................................................................................................................... 14 REFERENCES ............................................................................................................................. 15 APPENDIX ................................................................................................................................... 18 iii INTRODUCTION Elevated blood pressure (hypertension) puts arteries to the test. When exposed to a higher-than-normal blood pressure, the cells of the artery rearrange themselves; lay down more collagen to strengthen the vessel wall (fibrosis); and increase cell number (hyperplasia) (Humphrey, 2021). This process, described as remodeling of the artery, is best recognized in two tunica of the vessel, the tunica media and to a lesser extent the tunica adventitia (Majesky & Weiser-Evans, 2022, Majesky et al., 2012, Michel et al., 2021, McGrath et al., 2005, Siow & Churchman, 2007, Coen et al., 2011). Remodeling is also recognized in the many species in which the vasculature has been studied, from the mouse to the human. Missing from this is the consideration that the tunica adiposa, equivalent to the perivascular adipose tissue (PVAT), may also remodel in hypertension. PVAT is best recognized for its ability to produce anticontractile substances (Xia & Li, 2017, Gollasch, 2017). PVAT responds to hypertension/cardiovascular disease with a loss of anticontractile function due to a lower production or effectiveness of substances that reduce contraction. These include nitric oxide, hydrogen sulfide, and adiponectin to name a few. This knowledge is important but is limited in consideration of the overall functions of PVAT. We have a long-term hypothesis that PVAT has the potential to mechanosense and mechanorespond. One could argue that the loss of anticontractile function in the face of hypertension is just one such example of mechanosensing and responding to a change in pressure. Presently, we turn to a different response of PVAT, that of extracellular matrix (ECM) remodeling in the face of hypertension. The media (Humphrey, 2021) and, to some extent, the adventitia (Majesky & Weiser- Evans, 2022, Majesky et al., 2012, Michel et al., 2021, McGrath et al., 2005, Siow & Churchman, 2007, Coen et al., 2011) remodel with/after development of an elevated blood pressure with elevation in ECM proteins that ultimately increase fibrosis of the artery. Here, we test the specific hypothesis that PVAT remodels its extracellular matrix to become more fibrotic, as measured by changes in genes recognized to play a role in this process. In this study, we compare the PVAT to the adventitia directly. This is because the adventitia serves as the basement for PVAT, its closest neighbor. Also, the adventitia is collagen rich, while PVAT is comparatively collagen poor in the normal state (Watts et al., 2021). The model used is the Dahl SS male rat made hypertensive by being fed a high fat (HF) diet from weaning (Beyer et al., 1 2012, Fernandes, et al., 2018). The thoracic aorta, which is exposed to the highest blood pressures in the body, is the arterial model. Use of this specific artery provided three benefits. First, the PVAT of this tissue is discrete to this artery; it is not shared with a vein. Second, the thoracic aorta is large enough such that the adventitia can be dissected off from the media. Third and finally, the individual tunicas of one thoracic aortic media and adventitia provide sufficient RNA such that a full plate of ECM genes can be run for each tissue. This allows for a powerful comparison between adventitia and PVAT within one animal. 2 Animals and Diet MATERIALS AND METHODS Dahl SS male rats, just weaned (3-4 weeks of age), were purchased from Charles River Laboratories (RRID:RGD_2308886; Kingston, NY, USA). Once received at MSU, rats were randomized to Control diet or HF diet and fed for either 8 (N=5 pairs) or 24 weeks (N= 5 pairs). A total of twenty (20) rats were used in this study. The experiments that result from animals on diet at these two time points were carried out at independent times. Rats are given a unique group number and ear clipped to distinguish one from another. Animals were group housed (2/3 per cage dedicated to one diet) on a 12-hour light/dark cycle at 22–25°C room temperature. The animals were housed with Heat Treated Aspen Hardwood Laboratory Bedding (Northeastern Products Corp., Warrensburg, NY USA). The Control diet was 10% kCal fat (lard) and 0.2% Na (Diet D12450J, Research Diets, New Brunswick, NJ, USA) while the High Fat (HF) diet was 60% kCal high fat (lard) and 0.3% Na (Diet D12492). Diets were well labelled in a freezer in the same room housing experimental rats. Food and water were available ad libitum. MSU animal care staff and study staff worked together to ensure that food was fresh and feeding of a specific diet was not interrupted during the course of experiments. Procedures using animals complied with National Institutes of Health “Guide for the Care and Use of Laboratory Animals” (2011). Procedures used were approved by the MSU Institutional Animal Care and Use Committee (PROTO202000009). Finally, this study was conducted with “Animal Research: Reporting of In Vivo Experiments “(ARRIVE) guidelines (essential 10 and recommended) in mind. Sphygmomanometry Blood pressure measurements of rats were made by sphygmomanometry in conscious, restrained and warmed (~ 37°C for 3-5 minutes) rats. The animal was placed in a restraint device designed to maintain body temperature and keep the animal calm. The entire measurement process was automated via a computer program that controls the apparatus (CODA High Throughput system, Kent Scientific, Torrington, CT, USA). The tail-cuff was inflated 15 times (to a pressure of ~ 250 mmHg with a slow deflation over a period of ~ 15 seconds) with thirty- second intervals between inflations. Blood pressure was obtained during each inflation cycle by a volume pressure recording sensor; the final reading was the average of ten inflations. While this system non-invasively measures systolic, diastolic, and mean arterial pressure, we report 3 only mean arterial pressure. Once the measures were done, animals were returned to their home cage. This measure was made at least once during the time on diet and the day before animals were taken for experimentation. Dissection Rats were given pentobarbital as a deep anesthetic (80 mg kg-1, ip). A bilateral pneumothorax was created prior to vessel dissection from the rat. The thoracic aorta was dissected from the aortic arch to the diaphragm. Tissue dissection took place under a stereomicroscope and in a Silastic®- coated dish filled with physiological salt solution (PSS) containing [mM: NaCl 130; KCl 4.7; KH2PO4 1.18; MgSO4 • 7H2O 1.17; NaHCO3 14.8; dextrose 5.5; CaNa2EDTA 0.03, CaCl2 1.6 (pH 7.2)]. The whole aorta was threaded onto a wire mounted in a black silastic-filled dish. One small ring (2 mm wide) containing all arterial tunica was dissected off for histology (below). The wire was then secured to the silastic and all PVAT dissected off from the whole of the mounted aorta; this became the PVAT sample for the RT2 Profiler Arrays. The aorta was then removed from the wire, cut longitudinally and pinned lumen-side down. The adventitia was scored with a scalpel and gently teased off the media. This became the adventitia sample for the RT2 Profiler Arrays. Histology Immediately upon dissection, the 2 mm rings of thoracic aorta were formalin (10%)-fixed and paraffin embedded by MSU Investigative Histopathology. Sections (8 micron thick) were cut and tissues stained standardly with Masson Trichrome Stain. Sections were photographed on a Nikon TE2000 inverted microscope using a Nikon Digital Sight DS-Qil camera and Nikon NIS Elements BR 4.6 software. Settings were determined using tissue without stain as a negative control and were used consistently across all images. RNA Isolation, Quantification and Purity Adventitia (9 - 34 mg from the 5 aortae) or thoracic aortic PVAT (48 - 93 mg) was homogenized in 2 mL Beadruptor tubes with 1.4 mm ceramic bead media (catalog # 19-645-3, Omni International, Kennesaw, GA, USA) and 900 mL Qiazol Lysis Reagent (from Universal Mini Kit) in an Omni Beadruptor (Omni International; speed = 5.65 m/s, time = 30 seconds, cycles = 2). Following disruption, RNA was isolated with the RNeasy Plus Universal Mini Kit (catalog # 73404, Qiagen, Germantown, MD, USA) according to manufacturer’s recommended protocol. RNA concentration and purity were determined using a Nanodrop 2000c (Thermo Scientific, Waltham, MA, USA). 4 cDNA Preparation The RT2 First Strand Kit (catalog # 330421, Qiagen, Germantown, MD, USA) was used according to manufacturer’s recommended protocol to reverse transcribe 0.5 mg RNA into cDNA in a SimpliAmp thermal cycler (Applied Biosystems, Waltham, MA, USA). Extracellular Matrix and Cell Adhesion RT2 Profiler Arrays A single array plate (RT² Profiler™ PCR Array Rat Extracellular Matrix & Adhesion Molecules; GeneGlobe ID - PARN-013Z, Qiagen, Germantown, MD, USA) was used for comparison of extracellular matrix genes between each tissue and types of tissue. Adventitia and PVAT samples for each animal were run on individual plates; these plates were run on the same day. In this way, the adventitia and PVAT of the same aorta could be best compared. The PCR conditions were as follows (run on Applied Biosystems Quant Studio 7 Flex system connected to a Dell Optiplex XE2 computer: 1 cycle of 95oC 10 minutes; 40 cycles of 95oC 15 seconds; 60oC 1 minute RT2 SYBR Green Master Mix (catalog # 330623, Qiagen, Germantown, MD, USA) was used for all SuperArray plates. As stated in the RT2 Profiler PCR Array Handbook, the threshold value is determined when CT PPC is 20 + 2. All plates in this study were set to the same value of 0.16. Data were exported (described below). Array Analysis The RT2 Profiler Array uses a Microsoft Excel-based file that allows for import of CT values from the real-time PCR machine. Data were gathered in separate Excel sheets for each of the following four comparison: 8 week Adventitia HF vs Control; 8 week PVAT HF vs Control; 24 week Adventitia HF vs Control; and 24 week PVAT HF vs Control. In each sheet, CT values were compared to the average of five (5) different reference genes: b-actin (Actb), beta-2- microglobulin (B2M), hypoxanthine phosphoribosyltransferase 1 (Hprt1), lactate dehydrogenase A (Ldha) and Ribosomal protein, large, P1 (Rplp1). The 2-DCT were calculated for the gene of interest where DCT is [CT value of the gene of interest – C T value of averaged housekeeping gene]. From this, the fold change of HF/control was calculated. These are the values reported in Supplemental Table 1. A fold change of 2.00 or above was considered biologically meaningful. Statistically, the p values were calculated based on a Student’s t-test of the replicate 2-DCT values 5 for each gene in the Control group and Treatment (HF) groups. Generation of Volcano Plots of Differential Gene Expression at 8 and 24 Weeks on Diet Volcano plots were used to highlight the upregulated and downregulated genes in each tissue. Plots were generated using the RT2 Profiler Array analysis Excel document. The plots were modified to highlight the gene names of the top differentially expressed genes in each tissue and timepoint, denoted by black dots. Construction of Color Plots to Visualize Fold Change The Cytoscape application (Shannon et al., 2003) was leveraged to visualize the genes that were upregulated with a fold-change (FC) > 2 with a color-intensity plot. A fold-change of 2.00 is represented by the lightest color, and as the fold-change increases the color darkens in turn. These values were used to create a quantitative Venn diagram of Differentially Expressed Genes (DEGs) at 24 weeks where the shape fill represents PVAT gene FC, and the shape outline is indicative of adventitia gene FC. Construction of Gene Regulatory Network The Cytoscape plugin iRegulon was employed to understand the regulatory interactions among the DEGs within PVAT and adventitia at week 8 and 24 on HF vs Control diet through the construction of a gene regulatory network. iRegulon predicts transcription factors (TFs) involved in regulating co-expressed gene sets through the ranking of enriched motifs using position weight matrices (PWM) (Janky et al., 2014). Mus musculus was used in analysis as species for both analyses as there was no rat species offered in the program. The minimum identity between orthologous genes was set to 0.05, while the maximum false discovery rate (FDR) on motif similarity was defined as 0.001. The gene regulatory network was constructed using a threshold of >4 normalized enrichment score (NES) to select TFs for analysis in both tissues at 8 and 24 weeks. Immunohistochemistry protocol for thrombospondin1 expression Sections made from aorta taken for Trichrome staining were used. These paraffin- embedded sections (8 micron) were cut, dewaxed, and taken through a standard protocol using a Rabbit Vector kit (Vector Laboratories, Burlingame, CA, USA). Tissue sections were incubated 24 hours with primary antibody against Thrombospondin1 (1:40, Cat # 18304-1-AP, Anti- rabbit). Sections of the superior vena cava were used as a positive control. The same concentration of primary and secondary antibody was used on positive control/aortic/vena cava 6 sections. Sections were developed according to manufacturer’s instructions using a DAB developing solution (Vector Laboratories, Burlingame, CA, USA) for one minute. Binding was observed as a dark brown/black precipitate. All slides were counterstained with Vector Hematoxylin for 20 seconds, with nuclei stained blue. Sections were dried, coverslipped and photographed on a Nikon TE2000 inverted microscope using MetaMorph® software. Autowhite balance was used for all images. When brightness/contrast of these brightfield images were taken, they were done to the whole image and similarly between the images with and without the primary antibody. Data Presentation Weights and blood pressures of rats are reported as the means + SEM for the N=5 for each group. Histological images shown are those originally taken microscopically or brightened/contrasted comparatively between the control and HF condition. Images were brightened/contrasted on the whole, never in part. One RT2 Profiler Array plate containing a sample of adventitia from a high-fat fed rat did not amplify and was not included in subsequent analyses, meaning that the HF adventitia samples represent an N=4 while all other samples represent N=5. The volcano plot presents the average of the log base 2-fold-change in HF/control in all five pairs on x-axis with log base 10 of the p value generated within the RT2 Profiler Array Excel file are on the y-axis. Genes 2-fold upregulated at both the 8 and 24 week time point in PVAT and adventitia are shown as color plots, bar scaled for fold change. Cytoscape generated the gene regulatory plots for transcription factors involved in regulating gene sets, again using the mean values for the HF/Con value for all five pairs. Statistical Analyses A one-way ANOVA followed by a Sidak’s multiple comparison was used to determine statistical differences between the 8 and 24 week weights and the 8 and 24 week blood pressures. Here, a p value of 0.05 or less was considered significant. Analysis of the RT2 Profiler PCR Array results were complete using an Excel template distributed by Qiagen. A Student’s t-test was used to calculate the p-values for of the replicate 2-DCT values for each gene in the Profiler Array for adventitia and PVAT at 8 weeks. 7 RESULTS Blood pressure and body weights of experimental rats at 8 and 24 weeks on diets Figure 1 shares the final weights (Fig. 1a) and mean arterial blood pressures (MABP; Fig. 1b) of animals after 8 and 24 weeks of being on Control or HF diet. The body mass of both Control and HF diet fed rats were greater at 24 weeks compared to their 8-week counterpart group. Moreover, the mass of the HF rat was greater than the Control at the 24 week but not the 8 week time. Similarly, the MABP of the 24 week HF diet fed rat was significantly greater than the paired Control diet fed rat. This was not the case at the 8 week time point in which the blood pressures were statistically similar (p = 0.082, Sidak’s multiple comparison test). Thus, aortic samples came from animals without (8 week) and with (24 week) elevated mass and blood pressure. Histology of the thoracic aorta from rats on Control or HF diet for 8 and 24 weeks The small ring of thoracic aorta that gave rise to the adventitia and PVAT samples used in the RT2 Profiler Array were saved for histology. Figure 2 shares the Masson Trichrome staining of an exemplar of each of the five pairs. Of specific importance are the two (2) tunica that were dissected for measure of ECM genes. First, the adventitia stained a brilliant blue, indicative of collagen, in the aorta from animals 8 weeks on diet. Adventitia became more diffuse and less brilliant/dense with age (24 weeks on diet). This staining was more vivid in the adventitia vs the PVAT at both 8 and 24 weeks of diet. Second, PVAT was composed primarily of fascia with densely packed/small brown adipocytes in the Control fed rat. Comparatively, the HF diet increased the size and placement of white adipocytes within brown pads. This was readily apparent in the aorta from the rat on the HF diet for 24 weeks. Moreover, collagen was clearly present in the PVAT of the Control and PVAT from the HF rats but less dense than in the adventitia. Comparison of PVAT to adventitial genes at 8- and 24-weeks of Control or HF diet The absolute and relative levels of expression of all genes and their associated p values are shared in Table 1. At the 8 week time point, changes in the adventitia far outnumbered those observed in PVAT. Specifically, no genes reached the 2-fold threshold for change in the PVAT while 29 did so in the adventitia of the same vessel. Figure 3a details these genes in volcano plot form with the top genes that changed to the greatest magnitude labelled. Thrombospondin1 (Thbs1) and collagen 8a1 (Col8a1) were the genes with the greatest magnitude of change in the 8 adventitia at this 8 week on diet time point. By contrast, PVAT had the greatest number of changes at the 24 week time point: 47 genes upregulated 2-fold vs the 3 observed in the adventitia (Fig. 3a). Here, too, the thrombospondin 1 gene Thbs1 was one of the top three most upregulated genes. It was accompanied by ADAM metallopeptidase with thrombospondin type 1 motif, 2 (Adamts2) and platelet/endothelial cell adhesion molecule 1 (PECAM1). Figure 4 depicts the relative magnitude of changes of gene transcription at the 8 (Fig. 4a) and 24 (Fig. 4b) week time point. Only the adventitial results are represented in figure 4a because of the lack of genes that changed over 2-fold in PVAT at the 8 week time point. Figure 6 depicts the significant staining for thrombospondin-1 protein in the adventitia and PVAT at the 8 week and 24 week time points. Transcriptional drivers of adventitial and PVAT gene changes The Cystoscape plugin iRegulon allows for tracking of gene changes to curated relationships with transcription factors. Figure 5 shares these results. For twenty-two (22) of the genes 2-fold upregulated in the adventitia from the 8 week HF fed Dahl SS rat, the transcription factors Adarb1, Fos, Gm10323, Smad3, E2f1, Bcl11a, Rreb1, Jazf1, and MAPK1 were identified. For thirty-five (35) of the genes 2-fold upregulated in the PVAT from the 24 week HF fed Dahl SS rat, Adarb1 was again identified as a potential transcription factor as well as Stat1, Bcl6b, Tsnax, Pax8 and Jun. 9 DISCUSSION This study was designed to test the hypothesis that PVAT is responsive to hypertension and likely remodels to assist the blood vessel in protection against higher pressures. We investigated both the PVAT and tunica adventitia of the thoracic aorta. This was done to compare directly how neighboring tissues respond to high fat diet (HFD)-induced hypertension through changes in expression of ECM-related genes, as well as compare PVAT to a tissue better understood in its remodeling in response to hypertension. Tissue remodeling occurs before and after hypertension Our data support that ECM remodeling occurs in the thoracic aortic adventitia of the Dahl SS male rat prior to the onset of hypertension, and in the PVAT after hypertension is established. This general finding is important because it supports the idea that PVAT may be significantly responsive to elevation of blood pressures as opposed to a change in diet. Similarly, the adventitia may be an exquisitely sensitive barometer of local environmental change while PVAT might restrain/retard overall vascular remodeling. Collagen genes are upregulated in both adventitia and PVAT, but at different times Collagen is a protein well recognized to be involved in tissue remodeling. The genes for several collagen isoforms changed notably. Specifically, the gene for collagen isoform 4a2, important to the basement membrane of the cell, was upregulated in the adventitia at 8 weeks. Col2a1, a fibrillar collagen (Bella & Hulmes, 2017), was upregulated with the HF diet in both tissues, but at different times. It was upregulated at 8 weeks of diet in the adventitia but at 24 weeks in PVAT. Finally, Col8a1, the gene for the non-fibrillar short chain collagen, was the most upregulated gene in the adventitia at 8 weeks and upregulated in the PVAT at 24 weeks (Shuttleworth, 1997). This finding is consistent with the upregulation of type 8 (VIII) collagen in diseases or injury involving vascular remodeling (Hansen et al., 2016, Lopes et al., 2013). Upregulation of Col8a1 during the early remodeling response in the adventitia highlights the sensitivity of this vascular layer. The adventitia may preemptively adapt its ECM to meet changes in diet or before the onset of hypertension. The finding that the adventitia did not exhibit significant further changes at the 24 weeks of diet (comparing HF to Control) while PVAT had marked changes to gene expression is consistent with the idea that remodeling may be complete in the adventitia at this time. 10 Thrombospondin-1 is upregulated in adventitia and PVAT The gene Thbs1, which encodes for the matricellular ECM protein thrombospondin-1 (TSP1) (Bornstein, 1995), was one of the most upregulated genes in the adventitia at 8 weeks, and PVAT at 24 weeks on HF diet. Indeed, TSP1 staining was significantly higher in the adventitia and PVAT vs the media regardless of diet or time point (Figure 6). Immunohistochemical staining, considered a qualitative measure, was carried out not to determine whether TSP1 staining became higher in magnitude in PVAT at 24 weeks after the HF vs the control diet, but rather to provide evidence that the change in Thbs1 gene expression was relevant because these tissues ultimately make TSP1. TSP1 is a regulator of diverse cellular processes. These include ECM organization, matrix metalloproteinase activity, cell adhesion and migration, and apoptosis to name a few (Murphy-Ullrich, 2019). TSP1 also regulates the cellular functions implicated in the ECM homeostasis that are disturbed in remodeling. More specifically, TSP1 regulates the TGF-β pathway by binding and activating latent TGF-β, where activated TGF- β increased myofibroblast differentiation, recruited inflammatory cells, stimulated new matrix deposition, and promoted angiogenesis (Zhang et al., 2020, Rosini et al., 2018, Schultz-Cherry, et al., 1995, Kellouche et al., 2007, Murphy-Ullrich & Poczatek, 2000, Schultz-Cherry et al., 1994). In addition, fibroblast homeostasis is regulated through the C-terminal domain of TSP1 binding to collagen 1 (Rosini et al., 2018). The upregulation of TSP-1 in the adventitia and PVAT supports that both are remodeling their ECM at different times, but through at least one similar pathway. Interference with this pathway would thus potentially reduce remodeling of both layers. Identifying potential drivers of gene changes in adventitia and PVAT during remodeling Identification of transcription factors and their respective gene network gives mechanistic insight into what drives disease progression. Using the DEGs (>2FC) of the adventitia and PVAT, a gene regulatory network was computed to identify potential upstream regulators responsible for the observed remodeling in the present study. Adarb1 (adenosine deaminase RNA specific B1) was the only transcription factor predicted to influence gene expression in both the adventitia at 8 weeks (6 target genes) and PVAT at 24 weeks (12 target genes). Interestingly, the genes Ncam2, Ctnna2, Syt1, Hapln1, and Col8a1 were identified as targets for Adarb1 in both tissues, but at different times. Adarb1 was not, however, connected to Thbs1 in either the adventitia or PVAT. Nonetheless, Adarb1 may be responsible for mediating the ECM 11 remodeling responses of both tissue types in this model, potentially identifying a target to ameliorate the observed changes. PVAT is biologically responsive to hypertension The present study supports that the thoracic aortic PVAT, a unique fat depot surrounding the vasculature, is sensitive to changes in blood pressure and responds through differential expression of ECM-related genes and in morphology. This finding is consistent with our hypothesis that PVAT remodels its ECM in response to an elevated (sustained) pressure. Here, remodeling may serve as an adaptive response during hypertension to protect the vasculature it surrounds. The impact of this remodeling would be reflected by increased arterial stiffness. PVAT, in a healthy aorta, reduces the overall stiffness of the aorta (Tuttle et al., 2022). PVAT itself has a measurably lower stiffness than the artery it surrounds. Collagen content of PVAT was linearly and positively correlated with the low-stress stiffness of the tissue (Tuttle et al., 2022). PVAT might restrain the overall remodeling of the vessel, though unavoidably remodeling itself in the face of persistently elevated blood pressure. To this point, the PVAT of the isolated thoracic aorta of the HF vs Con diet fed male Dahl SS rats lost the ability to assist in arterial stress relaxation and displayed a modest increase in PVAT fibrosis (Watts et al., 2021). Limitations We recognize limitations to the present study. First, only male Dahl SS rats were used in the current study. In future studies, examining the response of female rats to HF diet induced hypertension in the present context is crucial. Second, study of the media was omitted in this study. Another limitation is that samples from HF or Control animals were only collected at week 8 (before hypertension onset) and 24 (after hypertension onset) for analysis. Other time points may lend insight into the progression of the ECM remodeling. The RT2 Profiler Array Kit that was used to determine gene expression has the innate limitation of being a finite, curated set of 84 genes related to the ECM. Those genes, however, are well curated and meaningful to the hypothesis at hand. We acknowledge the lack of statistical significance in the DEGs from Control to HF diet animals in both tissue types and timepoints. Despite having a relatively low number of genes reaching statistical significance, the magnitude of genes that had FC >2 at both timepoints may support that there are overall cumulative changes in the gene expression that could harmoniously contribute to ECM remodeling in the face of elevated blood pressure. While we discuss PVAT and adventitia as independent tunica, growing evidence 12 strengthens the existence of communication between PVAT and adventitia. For example, the PVAT-derived secretory protein complement 3 induced adventitial fibroblast migration and differentiation to contribute to the adventitial thickening and remodeling in the deoxycorticosterone acetate-salt hypertensive rat model (Ruan et al., 2010). Similarly, adventitial remodeling was promoted by dysfunctional PVAT in the obese mini pig model through the nod-like receptor protein 3 (NLRP3)/Interleukin-1 pathway (Zhu et al., 2019). Communication may occur/mediators may be passed between adventitia and PVAT during onset of hypertension that could mediate the changes in ECM gene expression that were observed in the current study. Tunica cross talk is not something we could measure in this specific study. 13 CONCLUSIONS PVAT of the thoracic aorta remodeled its extracellular matrix as a response to elevated blood pressure but not directly to diet. PVAT did not demonstrate biologically relevant changes in ECM genes until an elevated blood pressure was established. 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Final body weights and MABP of male Dahl SS rats after 8 or 24 weeks on diet Final weights of animals (grams) on control or HF diet (a), where the HF diet group had elevated body mass compared to control after 24 weeks on diet. MABP was determined before euthanasia of animals (b), demonstrating that animals after 8 weeks on either diet were normotensive. Animals on 24 weeks of HF diet had statistically elevated blood pressure compared to control. N=5 for all groups. HF=high-fat, MABP=mean arterial blood pressure. *= statistical difference (p<0.05) between control and high-fat fed animals. 18 Figure 2. Remodeling of adventitia and PVAT with progression of high-fat diet induced hypertension Representative images at 10X objective of male Dahl SS rat thoracic aorta and PVAT stained with Masson Trichrome to visualize collagen in blue after 8 (left) or 24 weeks (right) on either control (top) or high-fat (bottom) diet. (Scale bar=100µm) P=perivascular adipose tissue, A=tunica adventitia, M=tunica media, L=lumen. 19 a. b. 8 Week 24 Week Figure 3. Visualization of results of RT2 Profiler Array with Volcano Plots Volcano plots depicting the differential expression of ECM-related genes in thoracic aortic PVAT and adventitia of male Dahl SS rats fed a HF diet. Black line represents a p-value of 0.05, any points above the line having p-value < 0.05 and below a p-value > 0.05. Purple lines representing fold-change or the magnitude of change from control to HF diet animal gene expression. N=5 for all PVAT groups. N=5 for Control adventitia group, N=5 for HF adventitia at 8 weeks, and N=4 for HF adventitia group at 24 weeks. 20 a. b. Figure 4. Visualization of results of RT2 Profiler Array with color plots Fold-change for each DEG is visualized using Cytoscape through the utilization of color to represent fold-change. After 8 weeks on HFD there were 29 genes with FC>2 in the adventitia and no DEGs in PVAT (a), where shape fill color is representative of FC. A Quantitative Venn Diagram (b) depicting DEGs of ECM-associated genes from control to high-fat diet after 24- week HFD in thoracic PVAT (47 genes) and adventitia (3 genes) tissues. Fold-change for PVAT is represented as shape fill color, while adventitia fold-change is represented as border color. N=5 for all PVAT groups. N=5 for Control adventitia group, N=5 for HF adventitia at 8 weeks, and N=4 for HF adventitia group at 24 weeks. 21 a. 8 Week Adventitial Transcriptional Drivers b. 24 Week PVAT Transcriptional Drivers Figure 5. Gene regulatory network may identify transcription factors responsible for remodeling The Cytoscape plugin iRegulon was used to predict regulatory TFs and construct a gene regulatory network. TFs were included in the analysis with NES >4. TFs are identified by light blue shape fill and a shape outline color matching arrow color. Arrows are directed from TFs to their respective predicted target. The gene regulatory network of the DEGs of the adventitia at 8 weeks (a) predicted 8 TFs regulating 22 out of 29 DEGs. In the DEGs of PVAT at 24 weeks (b), 35 genes out of 47 were predicted to be regulated by 6 TFs. The TF Adarb1 was identified in both gene regulatory networks. N=5 for all PVAT groups. N=5 for Control adventitia group, N=5 for HF adventitia at 8 weeks, and N=4 for HF adventitia group at 24 weeks. DEG=Differentially expressed gene, NES=Normalized enrichment score, TF=Transcription factor. 22 Figure 6. Thrombospondin-1 protein is resident in both the Adventitia and PVAT in the thoracic aorta Representative images of thrombospondin-1 staining in thoracic aorta + PVAT in the Dahl SS male rat on 8 (left column) or 24 (right column) weeks of either control (top row) or HF (2nd row) of diet. L = lumen, A = adventitia, P = PVAT. The Vena Cava was used as a positive control. Arrows indicate region of interest. Inset box is image of tissue without primary antibody. Representative of four (4) of the 5 groups of rats used in this study. Horizontal bar = 100 micrometers. 23 Dahl SS Thoracic Aorta8 week24 weekControl DietHF DietVena CavaAPLAPLLLAAPPMMTSP-1No PrimaryPositive Conrol Table 1. The absolute and relative levels of expression for ECM-related genes in Adventitia and PVAT after 8 or 24 weeks on diet HF/CON 8 Week Adventitia PVAT HF/CON 24 Week Adventitia PVAT 0.47 0.208551 1.23 0.312363 1.32 0.844702 2.12 0.236062 BOLDED = >2 fold change Gene Identification Gene Abbrevia tion Adamts1 ADAM metallopeptidase with thrombospondin type 1 motif, 1 Adamts2 ADAM 0.83 0.503743 0.61 0.195847 0.85 0.46775 2.8 0.041484 metallopeptidase with thrombospondin type 1 motif, 2 Adamts5 ADAM 0.92 0.842989 1.8 0.084772 1.6 0.199426 1.16 0.841614 metallopeptidase with thrombospondin type 1 motif, 5 Adamts8 ADAM 1.63 0.505745 0.78 0.514198 2.19 0.192675 2.28 0.139635 metallopeptidase with thrombospondin type 1 motif, 8 Catenin (cadherin associated protein), alpha 1 Cd44 molecule Cadherin 1 Cadherin 2 Cadherin 3 Cadherin 4 Catna1 Cd44 Cdh1 Cdh2 Cdh3 Cdh4 Cntn1 Contactin 1 Col1a1 Col2a1 Col3a1 Col4a1 Col4a2 Col4a3 Col5a1 Col6a1 Collagen, type I, alpha 1 Collagen, type II, alpha 1 Collagen, type III, alpha 1 Collagen, type IV, alpha 1 Collagen, type IV, alpha 2 Collagen, type IV, alpha 3 Collagen, type V, alpha 1 Collagen, type VI, alpha 1 0.96 0.957035 1.23 0.140012 0.54 0.125818 1.08 0.748871 2.25 2.25 2.24 2.25 2.25 1.27 0.76 0.426544 0.78 0.506488 1.81 0.416544 2.28 0.139635 0.426544 0.78 0.514198 1.88 0.4125 2.28 0.139635 0.338308 1.3 0.980107 1.8 0.283664 2 0.106248 0.426544 0.78 0.514198 1.88 0.4125 2.28 0.139635 0.426544 0.78 0.514198 1.88 0.4125 2.28 0.139635 0.7154 0.86 0.59825 1.45 0.477198 2.28 0.116186 0.390571 1.43 0.122342 0.62 0.196201 1.15 0.618375 2.25 0.426544 0.78 0.514198 1.88 0.4125 2.28 0.139635 1.25 0.225276 1.17 0.283141 0.62 0.241327 0.88 0.710414 1.01 0.927021 0.89 0.118092 0.82 0.997707 0.83 0.456265 2.25 0.236822 0.95 0.780944 1.39 0.303401 1.14 0.907718 1.23 0.421439 0.62 0.222243 1.64 0.40041 1.99 0.102722 0.7 0.129678 1.95 0.003787 0.64 0.142817 1.09 0.99898 0.67 0.308425 0.9 0.557095 0.98 0.77797 1.07 0.856192 24 Table 1 (cont’d) Col8a1 Collagen, type VIII, alpha 1 Connective tissue growth factor Catenin (cadherin associated protein), alpha 2 Catenin (cadherin associated protein), beta 1 Extracellular matrix protein 1 Elastin microfibril interfacer 1 Ectonucleoside triphosphate diphosphohydrolase 1 Fibulin 1 Fibronectin 1 Hyaluronan and proteoglycan link protein 1 Intercellular adhesion molecule 1 Integrin, alpha 2 Integrin, alpha 3 Integrin, alpha 4 Integrin, alpha 5 (fibronectin receptor, alpha polypeptide) Integrin, alpha D Integrin, alpha E Integrin, alpha L Ctgf Ctnna2 Ctnnb1 Ecm1 Emilin1 Entpd1 Fbln1 Fn1 Hapln1 Icam1 Itga2 Itga3 Itga4 Itga5 Itgad Itgae Itgal Itgam Integrin, alpha M Itgav Itgb1 Itgb2 Itgb3 Itgb4 Lama1 Lama2 Lama3 Lamb2 Lamb3 Lamc1 Integrin, alpha V Integrin, beta 1 Integrin, beta 2 Integrin, beta 3 Integrin, beta 4 Laminin, alpha 1 Laminin, alpha 2 Laminin, alpha 3 Laminin, beta 2 Laminin, beta 3 Laminin, gamma 1 3.03 0.138332 0.78 0.514198 1.81 0.172648 2.28 0.139635 1.33 0.408371 0.96 0.980722 1.15 0.41725 1.59 0.324376 2.25 0.426544 0.78 0.514198 1.88 0.4125 2.28 0.13252 1.25 0.408053 1.32 0.396422 0.97 0.871093 0.85 0.605243 1.05 0.879192 0.95 0.721547 0.91 0.483367 1.81 0.15331 1.78 0.480747 0.78 0.514198 0.97 0.999962 2.28 0.117117 0.89 0.961393 0.62 0.173615 1.03 0.895562 2 0.061025 0.72 1.17 2.25 0.3879 0.99 0.92065 0.62 0.207833 1.45 0.261916 0.666907 1.22 0.52408 0.75 0.916293 1.13 0.662841 0.426544 0.78 0.514198 1.88 0.4125 2.28 0.139635 1.59 0.073663 1.33 0.395443 1.57 0.64529 2.01 0.096517 1.46 1.73 1.7 0.598119 0.97 0.762536 1.88 0.4125 2.28 0.139635 0.490205 0.89 0.774434 2.17 0.318011 2.28 0.118575 0.582493 0.87 0.66432 1.88 0.4125 2.02 0.081293 1.44 0.566926 0.78 0.514198 1.62 0.40785 2.28 0.120666 2.25 2.21 2.25 2.25 1.54 1.37 0.82 1.58 2.25 2.25 1.33 1.3 1.43 2.25 0.85 0.426544 1.39 0.460784 1.88 0.4125 2.2 0.149405 0.430728 0.78 0.514198 1.88 0.4125 2.28 0.139635 0.426544 0.95 0.812148 1.88 0.4125 2.28 0.104941 0.426544 0.78 0.514198 1.88 0.4125 2.28 0.139635 0.141375 1.24 0.372944 0.99 0.842516 1.09 0.752366 0.167628 0.93 0.491594 0.82 0.767487 1.19 0.472856 0.389657 1.56 0.109204 1.58 0.442458 1.79 0.073685 0.462275 0.78 0.514198 1.43 0.494551 2.17 0.092941 0.426544 0.78 0.514198 1.75 0.420651 2.28 0.128987 0.426544 0.78 0.514198 1.88 0.4125 2.28 0.139635 0.208304 1 0.938021 0.94 0.735998 1.28 0.430791 0.825973 0.8 0.543069 1.48 0.625865 2.28 0.111303 0.144247 0.95 0.6352 0.89 0.84472 1.49 0.410013 0.426544 0.78 0.514198 1.71 0.425105 1.83 0.168345 0.405646 1.01 0.956764 1.28 0.380409 1 0.937069 25 Table 1 (cont’d) Mmp10 Matrix metallopeptidase 10 2.25 0.426544 0.78 0.514198 1.88 0.4125 2.28 0.139635 Mmp11 Matrix 2.25 0.426544 0.86 0.696912 1.61 0.438479 2.11 0.082244 metallopeptidase 11 Mmp12 Matrix 0.97 0.460524 0.68 0.254984 1.53 0.45404 2.28 0.139635 metallopeptidase 12 Mmp13 Matrix 2.25 0.426544 0.76 0.482177 1.88 0.4125 2.28 0.139635 metallopeptidase 13 Mmp14 Matrix 1.28 0.395715 0.71 0.359091 1.02 0.779647 1.76 0.07832 metallopeptidase 14 (membrane-inserted) Mmp15 Matrix 2.25 0.426544 0.78 0.514198 1.88 0.4125 2.28 0.131325 metallopeptidase 15 Mmp16 Matrix 1.68 0.469895 0.78 0.514198 1.84 0.414523 2.28 0.139635 Mmp1 Mmp2 Mmp3 Mmp7 Mmp8 Mmp9 Ncam1 Ncam2 Pecam1 Postn Sele Sell Selp Sgce Sparc Spock1 Spp1 metallopeptidase 16 Matrix metallopeptidase 1a (interstitial collagenase) Matrix metallopeptidase 2 Matrix metallopeptidase 3 Matrix metallopeptidase 7 Matrix metallopeptidase 8 Matrix metallopeptidase 9 Neural cell adhesion molecule 1 Neural cell adhesion molecule 2 Platelet/endothelial cell adhesion molecule 1 Periostin, osteoblast specific factor Selectin E Selectin L Selectin P Sarcoglycan, epsilon Secreted protein, acidic, cysteine-rich (osteonectin) Sparc/osteonectin, cwcv and kazal-like domains proteoglycan (testican) 1 Secreted phosphoprotein 1 2.25 0.426544 0.78 0.514198 1.88 0.4125 2.28 0.139635 1.12 0.507937 0.73 0.296872 0.75 0.361293 1.13 0.750811 0.67 0.264197 0.78 0.514198 1.88 0.4125 2.28 0.139635 2.25 0.426544 0.78 0.514198 1.88 0.4125 2.28 0.139635 2.25 0.426544 0.78 0.514198 1.88 0.4125 2.28 0.139635 2.25 0.426544 0.78 0.514198 1.88 0.4125 2.28 0.139635 1.75 0.04871 0.69 0.361512 1.26 0.568614 2.28 0.075813 2.25 0.426544 0.89 0.650328 1.88 0.4125 2.28 0.064482 1.51 0.172947 1.54 0.419521 1.62 0.188309 2.62 0.170147 1.71 0.193679 1.13 0.094298 0.97 0.880192 1.29 0.577475 0.93 2.25 1.67 0.83 0.98 0.643118 0.79 0.500679 1.34 0.447935 2.27 0.115479 0.426544 1.16 0.451046 1.88 0.4125 2.28 0.076477 0.516275 0.78 0.514198 1.77 0.419518 2.28 0.139635 0.333458 0.99 0.980541 1.33 0.303203 1.41 0.560502 0.879272 1.29 0.1833 1.09 0.646491 1.01 0.915467 1.97 0.439024 0.97 0.746709 1.77 0.419064 2.23 0.098979 1.19 0.455051 1.32 0.400209 0.64 0.44197 1.87 0.141288 26 Table 1 (cont’d) Syt1 Synaptotagmin I Tgfbi Thbs1 Thbs2 Timp1 Timp2 Timp3 Tnc Vcam1 Vcan Vtn Transforming growth factor, beta induced Thrombospondin 1 Thrombospondin 2 TIMP metallopeptidase inhibitor 1 TIMP metallopeptidase inhibitor 2 TIMP metallopeptidase inhibitor 3 Tenascin C Vascular cell adhesion molecule 1 Versican Vitronectin 2.25 0.426544 0.77 0.600322 1.88 0.4125 2.28 0.609659 0.85 0.714402 0.78 0.514198 1.04 0.767841 1.86 0.140801 2.42 0.75 1.41 0.279098 0.88 0.608742 1.69 0.426543 2.35 0.05606 0.833778 1.42 0.240028 1.41 0.364759 1.96 0.117764 0.106101 1.02 0.921845 1.19 0.53468 1.14 0.752701 0.94 0.934397 1.06 0.537585 0.81 0.427482 1.19 0.526528 1.82 0.022644 0.54 0.006269 0.88 0.978859 0.94 0.709963 1.47 1.39 0.94 0.81 0.303013 1.27 0.662127 1.17 0.779541 1.97 0.107988 0.391319 0.74 0.422494 0.95 0.799497 1.66 0.232239 0.868758 0.87 0.470667 1.14 0.838404 2.28 0.069843 0.424098 1.16 0.848173 2.36 0.021202 1.2 0.392269 27