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Disturbed Flow Promotes Arterial Stiffening Through Thrombospondin-1

Originally publishedhttps://doi.org/10.1161/CIRCULATIONAHA.116.026361Circulation. 2017;136:1217–1232

Abstract

Background:

Arterial stiffness and wall shear stress are powerful determinants of cardiovascular health, and arterial stiffness is associated with increased cardiovascular mortality. Low and oscillatory wall shear stress, termed disturbed flow (d-flow), promotes atherosclerotic arterial remodeling, but the relationship between d-flow and arterial stiffness is not well understood. The objective of this study was to define the role of d-flow on arterial stiffening and discover the relevant signaling pathways by which d-flow stiffens arteries.

Methods:

D-flow was induced in the carotid arteries of young and old mice of both sexes. Arterial stiffness was quantified ex vivo with cylindrical biaxial mechanical testing and in vivo from duplex ultrasound and compared with unmanipulated carotid arteries from 80-week-old mice. Gene expression and pathway analysis was performed on endothelial cell–enriched RNA and validated by immunohistochemistry. In vitro testing of signaling pathways was performed under oscillatory and laminar wall shear stress conditions. Human arteries from regions of d-flow and stable flow were tested ex vivo to validate critical results from the animal model.

Results:

D-flow induced arterial stiffening through collagen deposition after partial carotid ligation, and the degree of stiffening was similar to that of unmanipulated carotid arteries from 80-week-old mice. Intimal gene pathway analyses identified transforming growth factor-β pathways as having a prominent role in this stiffened arterial response, but this was attributable to thrombospondin-1 (TSP-1) stimulation of profibrotic genes and not changes to transforming growth factor-β. In vitro and in vivo testing under d-flow conditions identified a possible role for TSP-1 activation of transforming growth factor-β in the upregulation of these genes. TSP-1 knockout animals had significantly less arterial stiffening in response to d-flow than wild-type carotid arteries. Human arteries exposed to d-flow had similar increases TSP-1 and collagen gene expression as seen in our model.

Conclusions:

TSP-1 has a critical role in shear-mediated arterial stiffening that is mediated in part through TSP-1’s activation of the profibrotic signaling pathways of transforming growth factor-β. Molecular targets in this pathway may lead to novel therapies to limit arterial stiffening and the progression of disease in arteries exposed to d-flow.

Introduction

Clinical Perspective

What Is New?

  • The development of a novel murine model of disturbed blood flow to stimulate arterial stiffening through collagen deposition in young male and female mice.

  • The authors discovered a critical role for thrombospondin-1 (TSP-1) in stimulating disturbed flow–induced arterial stiffening, which was significantly attenuated in the TSP-1 knockout animal.

  • Blockade of TSP-1 activation of transforming growth factor-β decreased the upregulation of profibrotic genes that contribute to arterial stiffening.

  • TSP-1 localized to regions of disturbed flow in arteries from patients with peripheral artery disease, and these arteries had similar increases in collagen gene expression.

What Are the Clinical Implications?

  • Existing literature has linked TSP-1 to peripheral artery disease, but the mechanism and biological importance of this relationship have not been shown.

  • This work validates the localization of increased TSP-1 to regions of disturbed flow in peripheral arteries, which may provide the mechanism by which TSP-1 is increased in peripheral artery disease.

  • This work links TSP-1 upregulation to arterial stiffening and identifies TSP-1 as an important promoter of pathological arterial remodeling in peripheral artery disease arteries under disturbed flow conditions.

  • The profibrotic molecular pathways stimulated by TSP-1 may provide novel pharmaceutical targets for promoting arterial health and inhibiting pathological arterial remodeling.

Arterial stiffening is a complex age-related remodeling process that independently doubles patient mortality and may contribute to the onset and progression of atherosclerotic cardiovascular disease.13 Material changes to the elastin and collagen content of the vessel wall mediate arterial stiffness.4 Mechanistically, transforming growth factor-β (TGF-β) has been implicated in arterial stiffening via profibrotic collagen deposition into the adventitia of the arterial wall during aging,5,6 but its role in this process appears more complicated than simple upregulation of TGF-β.7 This is not surprising because TGF-β activation and signaling are complex, regulated by TGF-β receptor expression and matricellular cues. Because the signaling pathways that initiate and sustain arterial stiffening are not well understood,8,9 there is a critical need to develop and test novel translational models of arterial stiffening.

Fluid shear stress is known to be an important mediator of arterial remodeling.10 In areas of disturbed flow (d-flow), low and oscillatory wall shear stress (WSS) stimulates proinflammatory responses from endothelial cells (ECs) and promotes atherosclerotic remodeling of affected arteries through lipid accumulation and elastin degradation.11,12 It is exciting that the EC response to d-flow can be used to discover novel shear-sensitive genes important to the regulation of vascular remodeling.13 One plausible pathway that links d-flow with TGF-β–mediated arterial stiffening is thrombospondin-1 (TSP-1), which we and others have identified as a shear-sensitive protein that is upregulated under d-flow conditions.13,14 TSP-1 activation of TGF-β is thought to be mediated through TSP-1 binding to TGF-β′ latent activating factor (LAF) (or peptide).15 It is important to note that this pathway can be modified by pharmacologically inhibiting TSP-1 binding to LAF via peptide inhibitors (eg, LSKL [Anaspec]).16,17 TSP-1 has also been implicated in dysregulation of blood flow, impaired vessel dilation, and increased vascular tone.18,19 However, these activities appear to be mainly mediated through its interaction with cell receptors CD36 and CD47 and guanylate cyclase pathways,20,21 as opposed to profibrotic signaling and collagen deposition characteristic of stiffened arteries.

Previous studies have shown a relationship between d-flow and arterial stiffening. D-flow induces extracellular matrix stiffening under diabetic conditions,22 and by MRI, d-flow portions of human coronary arteries are stiffer than stable flow (s-flow) locations.23 However, the causal link between d-flow and arterial stiffening has not been established. Thus, the objective of this study was to determine whether d-flow promoted arterial stiffness in the absence of atherosclerosis, and then to discover the critical signaling pathways involved. We hypothesize that d-flow will promote arterial stiffening through TSP-1 upregulation. Here, we test arterial stiffening ex vivo by cylindrical biaxial testing and in vivo by strain analysis. The reversibility of this pathway is tested by disrupting TSP-1 activation of TGF-β with LSKL. Finally, the effect of d-flow on arterial stiffening is tested in a TSP-1 knockout (KO) mouse, and human arteries exposed to d-flow are used to validate the linking of d-flow with TSP-1 expression and profibrotic signaling pathways.

Methods

Partial Carotid Ligation Model of D-Flow

Partial carotid ligation studies24 were performed on young and old (12- to 20-week-old; 80-week-old) mice (C57BL/6×129/SvEv [S129], C57BL/6J [C57] [Jackson Laboratory]; TSP-1 KO [C57Bl/6×129S2/SvPas] [Jackson Laboratory]) as approved by Emory University’s Institutional Animal Care and Use Committee. Initial mechanical characterization was performed on young male and female S129 mice. Figure I in the online-only Data Supplement provides an overview of experimental methodology and time points.

Quantification of WSS Values

The in vivo hemodynamic environment was characterized in the mouse carotid arteries and human peripheral arteries by using ultrasound data. To describe the WSS disturbances in arteries with and without inline flow, a representative patient’s duplex ultrasound data were analyzed. Here, the superficial femoral artery represented s-flow (above occlusion=unimpeded inline flow) and the posterior tibial artery (below occlusion where flow is not inline but enters the vessel via collateral pathways) represented d-flow. Temporal WSS waveforms were quantified by Equation 1:

Mechanical Testing of Arterial Stiffness and Biochemical Analysis of Arteries

Murine common carotid arteries underwent cylindrical biaxial biomechanical testing with collagen and elastin quantification as published.25,26 An unpaired, 2-tailed t test was used to compare mean values with statistical significance corrected for multiple comparisons (P<0.0031 for compliance curves and P<0.0029; this is signified in the figures by §).

EC-Enriched RNA Isolation from Carotid Arteries

Total RNA from intima was collected from the left carotid artery (LCA) and right carotid artery (RCA) at designated time points as published.24 EC-enriched RNA was then tested for TSP-1, TGF-β1 to 3, TGF-β receptors 1 to 3, TSP-1 receptors CD36 and CD47, profibrotic genes (connective tissue growth factor [CTGF] and plasminogen activator inhibitor-1 [PAI1]), and collagen genes (Col1a1, Col4a1, and Col16a1). We used the naturally occurring differences in WSS between the greater (s-flow) and lesser (d-flow) curvature of the aortic arch to test for TSP-1 expression in response to chronic d-flow. Paired or unpaired 2-tailed t tests were used to compare mean values as appropriate with statistical significance set at P<0.05.

Microarray Procedure, Data Analysis, and Bioinformatics

EC-enriched RNA from 9 LCAs and 9 RCAs were pooled for microarray analysis (n=3 with a total of 9 mice used per each group). RNA sample quality was confirmed, and the normalized microarray data were screened for statistically significant genes that were differentially expressed (LCA versus RCA) with >1.5-fold increase.13 Pathway enrichment analysis was performed by mapping these differentially expressed genes to GeneGO’s MetaCore. This program functions as an integrated software suite for functional analysis of signaling pathways. GeneGO pathways were enriched by the 0.01 gene list derived from the EC-enriched microarray data. The gene array data along with the metadata were uploaded to the GEO Repository at the National Center for Biotechnology Information (Accession No. GSE87199).

Quantitative Polymerase Chain Reaction Validation

Total RNA of each sample was transcribed into cDNA,24 and quantitative polymerase chain reaction results were normalized for 18S RNA expression in each sample.13 Human artery samples were collected from amputated limbs after informed consent under an institutional review board–approved protocol (Emory University–approved institutional review board protocol No. 51432). Arterial specimens were segregated before testing into d-flow and s-flow conditions depending on the presence or absence of inline flow to the artery. All d-flow arteries (n=6) were in patients with proximal inflow occlusion and distal reconstitution through collateral pathways. All s-flow arteries (n=4) had inline flow without obstructive peripheral artery disease (PAD). An unpaired 2-tailed t test was used to compare mean values with statistical significance at P<0.05.

Histological and Immunohistochemical Analysis

Arteries were stained for histological analyses, and myointimal hyperplasia was quantified in a blinded fashion as published.27 Elastin architecture was visualized by autofluorescence. Immunohistochemistry was performed as published.13,24 An unpaired 2-tailed t test was used to compare mean values with statistical significance at P<0.05.

In Vitro EC Response to Oscillatory and Laminar WSS

Human aortic ECs were commercially obtained (Genlantis) and grown to confluence on 10-cm petri dishes. They were then exposed to oscillatory shear (±5 dyne/cm2 at 1 Hz) to mimic d-flow or laminar shear (15 dyne/cm2) to mimic s-flow. Static culture served as a no-shear control. After 48 hours, human aortic EC RNA was collected and analyzed. Multiple comparisons between treatment groups were statistically tested with analysis of variance with the Tukey post hoc test performed for statistical significance of P<0.05.

TSP-1 activation of TGF-β is mediated by LAF.28 To test the role of TSP-1 on TGF-β activation, we used 5 μmol/L LSKL as a competitive inhibitor of LAF and equimolar SLLK (Anaspec) as a nonsense peptide control. For in vivo testing, LSKL or SLLK was administered 4 mg/kg intraperitoneally every other day as published.29 Intraperitoneal treatments began 5 days before partial carotid ligation and were continued through the terminal end point of 3 days. Paired or unpaired 2-tailed t tests were used to compare mean values as appropriate with statistical significance set at P<0.05.

In Vivo Strain Measurements

Serial B-mode ultrasound data were acquired in the mid-LCA and -RCA at 3 days and 1, 2, and 4 weeks post–partial carotid artery ligation (n=9). The lumen boundaries were manually segmented in the middle 3 mm of each vessel, at least 1 mm proximal to the carotid bifurcation, across 1 cardiac cycle (≈60 images). A spline was fit to the segmented contours, and diameter values were quantified at 10 equally spaced intervals. The resulting diameter waveforms were averaged, generating a global diameter waveform for the vessel, and the circumferential deformation (ie, change in diameter) across the cardiac cycle was quantified. The maximum circumferential (Green) strain (), was calculated as a surrogate for arterial stiffness for each vessel. An unpaired 2-tailed t test was used to compare mean values with statistical significance of P<0.05.

Statistical Analysis

Statistical analyses were performed by using Graph-Pad Prism and Microsoft Excel statistical packages. For in vitro polymerase chain reaction testing, analysis of variance with Tukey post hoc analysis was performed. For single comparisons, the paired or unpaired 2-tailed Student t test, with significance set at P<0.05, was used (*P<0.05; †P<0.01; ‡P<0.001). Compliance and pressure diameter measurements were tested by unpaired 2-tailed Student t tests with statistical significance corrected for multiple comparisons (§P<0.0031 and §P<0.0029, respectively).

Results

Partial Carotid Ligation Leads to D-Flow in Young Mouse Arteries

Partial carotid ligation induces d-flow (Figure 1A and 1B) in 12-week-old S129 mice similar to that published in ApoE–/– mice.24. EC purity of intimal RNA was determined by quantitative polymerase chain reaction for platelet EC adhesion molecule (an EC marker), α-SMA (a smooth muscle cell marker), and CD11b (a leukocyte marker) (Figure 1C, left and middle). D-flow parameters were internally validated by quantifying the EC response to partial carotid ligation via downregulation of well-known shear-sensitive genes (KLK10; KLF2) in the LCA (Figure 1C, right). We then modeled WSS at the initial time point (24 hours) and out to the latest time point of these studies (6 weeks). WSS in the RCA demonstrated physiological WSS or s-flow out to 6 weeks post–partial ligation of the LCA. In contrast, the LCA demonstrated low and oscillatory WSS (d-flow) early and persistently low WSS out to 6 weeks after partial carotid ligation (Figure 1D).

Figure 1.

Figure 1. Partial carotid ligation invokes disturbed flow to the intima and downregulates shear-sensitive genes.A, Carotid artery duplex ultrasound demonstrates flow reversal and depressed velocities in the left carotid artery (LCA) that underwent partial ligation. Right carotid artery (RCA) was not manipulated and demonstrates unidirectional flow. (Note differences in the scale bars on the y axis [LCA: –50, 0, +50 mm/s; RCA: –150, 0, +150 mm/s at 1 day postligation and –200, 0, +200 mm/s at 42 days postligation, respectively]). B, Using velocity data points from the ultrasound images, the LCA had significantly diminished flow rates in comparison with the RCA through 6 weeks post–partial carotid ligation. C, Left, Intimal RNA demonstrates EC enrichment by the selective and segregated PECAM-1 expression from that of α-SMA or CD11b at 24 hours. Middle, RNA from the media/adventitia of these arteries demonstrates increased α-SMA. Right, D-flow conditions were then internally validated 24 hours after partial ligation by the significant downregulation of the shear-sensitive genes KLK10 and KLF2 in the LCA. n=3; mean±SEM; unpaired Student t test with significance at P<0.05. D, WSS in the RCA is consistent with stable flow (s-flow) throughout the experiment. However, the LCA manifests disturbed flow (d-flow) with low and oscillatory WSS acutely and low WSS throughout the latest time point of experiments in this work (6 weeks). *P<0.05. †P <0.01. ‡P<0.001. EC indicates endothelial cell; PECAM-1, platelet endothelial cell adhesion molecule; α-SMA, α-smooth muscle cell; and WSS, wall shear stress.

D-Flow Causes Arterial Stiffening and Increased Collagen Deposition

Twelve-week-old S129 mice underwent partial carotid ligation to induce d-flow in the LCA. After 4 weeks of d-flow, LCAs demonstrated significant arterial stiffening in comparison with RCAs by pressure diameter and compliance curves generated from ex vivo cylindrical biaxial testing (Figure 2A and 2B). Stiffened arterial remodeling was induced by d-flow in both male and female mice and over a range of 12 to 20 weeks (Figures II and III in the online-only Data Supplement). To compare the arterial stiffening induced by d-flow in our model with that which occurs naturally with aging, we measured the arterial stiffening occurring in response to d-flow in 12-week-old mice and unmanipulated carotid arteries of 80-week-old mice. Despite the 80-week carotid arteries having a larger diameter than the young LCAs yielding differences at higher pressures in the pressure diameter curve (Figure 2C), the compliance in the unmanipulated aged carotid arteries was remarkably similar to that of young arteries under d-flow (Figure 2D). To examine the material cause of stiffening under d-flow in our model, the collagen and elastin content were quantified. Collagen content in the LCA was increased within 2 weeks of d-flow (Figure IIIE in the online-only Data Supplement) and significantly increased over that of the RCA at 4 weeks, whereas elastin content was not affected (Figure 2E).

Figure 2.

Figure 2. D-flow and aging stiffen arteries similarly.A and B, Pressure diameter and compliance curves demonstrate that the carotid arteries of 12-week-old animals who had their LCA exposed to d-flow () were significantly stiffer than the contralateral RCA (○), which were not manipulated and exposed to s-flow. C and D, We then compared carotid arteries from 12-week-old mice exposed to d-flow with unmanipulated (s-flow) carotid arteries from aged (80-week-old) animals. Because of the enlarged diameter of the older arteries, the pressure diameter curve of the aged carotid arteries demonstrated greater dilation than the 12-week-old mice exposed to d-flow beginning at 60 mm Hg. However, the compliance curves were very similar without any statistically significant differences in compliance between the 12-week-old d-flow carotid arteries and those of the unmanipulated aged murine carotid arteries. n=6; mean±SEM; unpaired Student t test with significance (§) at P<0.0029 and P<0.0031, respectively. E, Structurally, the arterial stiffening from d-flow was attributable to increased collagen deposition, which was found in the LCA (d-flow) in comparison with RCA (s-flow), and not attributable to changes in elastin, which were similar between groups. n=12 RCA; n=14 LCA; mean±SEM; unpaired Student t test with significance set at P<0.05. *P<0.05. †P <0.01. ‡P <0.001. DAPI indicates 4′,6-diamidino-2-phenylindole; d-flow, disturbed flow; EC, endothelial cell; LCA, left carotid artery; RCA, right carotid artery; s-flow, stable flow; and TSP-1, thrombospondin-1.

Pathway Analysis Identified Prominent Role for TGF-β Pathways in D-Flow Model of Arterial Stiffening

To determine potential mechanisms by which d-flow causes arterial stiffening, we performed gene array studies by using EC-enriched RNA. Differentially expressed genes from the endothelial-enriched RNA samples obtained from the intima of d-flow LCA and s-flow RCA underwent pathway analysis with the use of MetaCore GeneGo software. Here we identified a prominent role for TGF-β pathways in our model (Table).

Table. Pathway Analysis of EC-Enriched RNA Under D-Flow Versus S-Flow Conditions

Sample No.PathwaysEnrichment Ratio (Genes Changed/Total Genes in the Pathway)False Discovery RateP Value
1Cytoskeleton remodeling; TGF, WNT, and cytoskeletal remodeling107/1112.4×10–222.71×10–25
2Cytoskeletal remodeling96/1025.6×10–181.6×10–20
3Endothelial to mesenchymal transition48/511.7×10–98.8×10–11
4TGF-β receptor signaling48/503.2×10–101.06×10–11
5Development TGF-β–dependent induction of EMT via MAPK46/471.6×10–103.05×10–12

Pathway analysis of EC-enriched RNA from murine carotid arteries exposed to disturbed flow demonstrates a prominent role for TGF-β pathways in this model. d-flow indicates disturbed flow; EC, endothelial cell; EMT, epithelial–mesenchymal transition; MAPK, mitogen-activated protein kinase; s-flow, stable flow; TGF-β, transforming growth factor-β; and WNT, Int/Wingless.

TSP-1 but not TGF-β Was Upregulated In Vivo by D-Flow

Next, we queried TGF-β/TGF-β receptor expression. It is surprising that TGF-β expression was not upregulated by d-flow in vivo, but TGF-β1 was initially decreased 24 hours after partial ligation in the EC-enriched LCA RNA (Figure 3A). There were not any other significant differences at 24 hours in TGF-β subtypes or TGF-β receptors in either the EC-enriched or media/adventitia RNA (Figure 3B through 3L).

Figure 3.

Figure 3. TGF-β and TGFBR expression in the carotid arterial wall in response to d-flow in vivo. A and D, TGF-β1 is downregulated in the endothelium (but not media/adventitia) in response to d-flow. B through F, TGF-β2 and TGF-β3 are not changed in the endothelium or media/adventitia under d-flow. G through L, TGFBR1 to 3 are not changed by d-flow in the endothelium or media/adventitia in vivo. n=3; mean±SEM; paired Student t test with significance set at P<0.05. *P<0.05. †P<0.01. ‡P<0.001. D-flow indicates disturbed flow; LCA, left carotid artery; RCA, right carotid artery; TGF-β, transforming growth factor-β; and TGFBR, TGF-β receptor.

However, there was a significant increase in TSP-1 in both the EC-enriched intimal RNA and the RNA from the media/adventitia in the d-flow LCAs versus s-flow RCAs (Figure 4A and 4B). Similarly, immunohistochemical staining of the carotid arteries demonstrated increased TSP-1 in the intima and whole artery of the LCA in comparison with RCA, validating the quantitative polymerase chain reaction expression (Figure 4C). To validate the role of chronic d-flow on TSP-1 upregulation, we used the lesser curvature and greater curvature of the aortic arch. Again, there was significantly greater expression of TSP-1 in the d-flow lesser curvature than in the s-flow greater curvature (Figure 4D and 4E). These results demonstrate that TSP-1 is upregulated by d-flow under both surgically induced acute changes in the carotid artery and chronic exposure in the lesser curvature of the aortic arch, where d-flow naturally occurs. The TSP-1 receptors CD36 and CD47 were not significantly elevated in the LCA at 24 hours (Figure 4F and 4G).

Figure 4.

Figure 4. Acute and chronic exposure to d-flow induces TSP-1 expression in vivo.A and B, TSP-1 expression was increased in the d-flow LCA vs s-flow RCA in the endothelium and media/adventitia. n=3; mean±SEM; paired Student t test; *P<0.05. C, TSP-1 staining (red: TSP-1; blue: DAPI) in the mouse carotid artery (L: lumen of artery) is increased under d-flow. C1 and C2, Cross-section IHC staining for TSP-1 in the RCA and LCA demonstrates increased TSP-1 in the LCA (d-flow) at day 3 post–partial carotid ligation. C3 and C4, Enface IHC staining in the endothelium of the RCA and LCA demonstrates increased TSP-1 in the LCA at day 3 post–partial carotid ligation. D and E, Increased TSP-1 expression in the endothelium and media/adventitia under chronic d-flow conditions in the lesser curvature (LC) in comparison with the greater curvature (GC) of the aortic arch, which is s-flow naturally. F and G, CD36 and CD47 expression were not significantly changed in the endothelium under d-flow conditions. n=4; mean±SEM; paired Student t test with significance set at P<0.05. Scale=20 μm. *P<0.05. †P<0.01. ‡P<0.001. DAPI indicates 4′,6-diamidino-2-phenylindole; d-flow, disturbed flow; IHC, immunohistochemical; LCA, left carotid artery; RCA, right carotid artery; and TSP-1, thrombospondin-1.

To better understand which genes were changed by d-flow over the 4 weeks of the experiment, we analyzed changes in gene expression at day 3 and weeks 1, 2, and 4. Here, we found TGF-β1 (decreased in the LCA at 24 hours) was increased in the LCA at 1 week (Figure 5A) without further differences in TGF-β1 to 3 (Figure 5A through 5C). Nor were there any significant differences in CD36 or CD47 expression identified (Figure 5D and 5E). However, there was sustained TSP-1 upregulation in the LCA that was significant at day 3 and week 1 (Figure 5F). Similarly, there was upregulation of the profibrotic genes in the LCAs with CTGF being statistically significant at 1 week, and PAI1 being significantly elevated at 3 days and 4 weeks (Figure 5G through 5I).

Figure 5.

Figure 5. Temporal gene changes in the endothelial RNA under d-flow.A through E, TGF-β1 is increased at 1 week in the LCA, but there were no other significant differences in TGF-β1 to 3, CD36 or CD47 identified over these time points (day 3, weeks 1, 2, and 4). F, TSP-1 was significantly increased in the LCA at day 3 and week 1 and demonstrated a trend toward significance at week 4. G, Col1a1 was not significantly increased at these time points. H and I, CTGF was significantly increased at 1 week, and PAI1 was significantly increased at day 3 and week 4. n=3 to 4; mean±SEM; unpaired Student t test with significance set at P<0.05. *P<0.05. †P<0.01; ‡P<0.001. CTGF indicates connective tissue growth factor; d-flow, disturbed flow; LCA, left carotid artery; PAI1, plasminogen activator inhibitor-1; RCA, right carotid artery; TGF-β, transforming growth factor-β; and TSP-1, thrombospondin-1.

Disrupting TSP-1 Activation of TGF-β by Inhibiting LAF

Given the prominent role of TGF-β pathways demonstrated in pathway analyses and the upregulation of TSP-1 by d-flow, we hypothesized that TSP-1 activation of TGF-β was a critical mediator of arterial stiffening in this model. In this pathway, TSP-1 binds to the LAF on TGF-β, leading to TGF-β activation (Figure 6A). To test the role of LAF on downstream profibrotic signaling in vitro, we mimicked d-flow with oscillatory shear and s-flow with laminar shear in human aortic ECs. We used LSKL to inhibit TSP-1 binding to TGF-βs LAF28; SLLK was the control peptide. Here LSKL treatment did not inhibit the oscillatory shear increase in TSP-1 or TGF-β1 in comparison with SLLK (Figure 6B and 6C), but LSKL did significantly decrease the elevations of the profibrotic TGFβ target genes Col1a1, CTGF, and PAI1 (Figure 6D through 6F) in comparison with the SLLK control under oscillatory shear. This supports a connection between increased TSP-1 expression and TGF-β activation in the upregulation of these profibrotic target genes.

Figure 6.

Figure 6. D-flow increases profibrotic gene expression in human aortic endothelial cells in vitro and LSKL downregulates these genes in vitro and in vivo. Differences between oscillatory shear (OS) and either static (ST) or laminar shear (LS) are demonstrated above ST and LS, whereas differences between OS+LSKL and OS+SLLK are listed above their respective columns. A, Putative mechanism by which TSP-1 can activate TGF-β and how the small peptide LSKL competitively inhibits TSP-1 activation of TGF-β. B, ECs under OS have increased TSP-1 expression in comparison with ECs under ST and LS conditions. TSP-1 expression is not affected by LSKL treatment or the control peptide (SLLK). C, TGF-β1 was also upregulated in ECs by OS and not affected by LSKL or SLLK. D, Col1a1 was upregulated by OS over ST and LS, and LSKL treatment significantly decreased Col1a1 expression under OS conditions in comparison with SLLK treatment. E, CTGF is significantly upregulated by OS (in comparison with ST and LS), and LSKL treatment significantly decreased CTGF expression under OS conditions in comparison with SLLK. F, PAI1 is significantly upregulated by OS (in comparison with ST and LS), and LSKL treatment significantly decreased PAI1 expression under OS conditions in comparison with SLLK. n=5 to 6; mean±SEM; analysis of variance with the Tukey post hoc test with significance set at P<0.05. *P<0.05. †P<0.01. ‡P<0.001. G through I, In vivo, LSKL globally decreased expression of profibrotic genes in comparison with SLLK control treatment in both the RCA (s-flow) and LCA (d-flow). Here only CTGF was still significantly upregulated in the LSKL treatment group by d-flow. J, TSP-1 expression the LCA of LSKL-treated animals was still significantly increased in comparison with the RCA of LSKL-treated animals. K through O, Again, there were no significant differences in TGF-β or TGFBR expression under d-flow or s-flow conditions (LCA vs RCA) in either the LSKL or SLLK groups. However, there were contrasting trends with greater TGF-β1 expression in SLLK-treated animals and greater TGF-βR1 to 3 expression in the LSKL-treated animals. P and Q, There were not any flow-mediated differences in the expression of CD36 or CD47 identified within groups, but there was a trend toward increased CD47 in the LSKL-treated animals in comparison with SLLK in both the LCA and RCA. n=3 to 4; mean±SEM; paired Student t test with significance set at P<0.05. *P<0.05. †P<0.01. ‡P<0.001. CTGF indicates connective tissue growth factor; d-flow, disturbed flow; EC, endothelial cell; HAEC, human aortic endothelial cell; LCA, left carotid artery; PAI1, plasminogen activator inhibitor-1; RCA, right carotid artery; TGF-β, transforming growth factor-β; TGFBR, TGF-β receptor; and TSP-1, thrombospondin-1.

We then tested the effectiveness of LSKL to decrease these genes in vivo. Here, LSKL globally decreased gene expression of these same profibrotic genes (Col1a1, CTGF, PAI1) in comparison with SLLK-treated mice in both the RCA and LCA (Figure 6G through 6I). Although decreased in comparison with LCA in SLLK animals, the LCA of LSKL-treated mice still had significant increases in TSP-1 and CTGF expression in comparison with that of LSKL RCA (Figure 6H and 6J). There were no differences demonstrated in either Col1a1 or Pai1 (Figure 6G and 6I). TGF-β1 to 2 and TGF-βR1 to 3 expression levels were not significantly different between RCA and LCA (Figure 6K through 6O). TGF-β3 was not sufficiently expressed for analyses in these groups. There was an interesting trend in TGF-β gene regulation with TGF-β1 trending upward in SLLK-treated animals and TGF-βR1-3 trending upward in LSKL-treated animals, suggesting a homeostatic interaction between LSKL treatments and TGF-β activity. However, these trends did not appear to be influenced by d-flow or s-flow conditions. Similarly, CD36 and CD47 were not significantly different between RCA and LCA in either group (Figure 6P through 6Q), but CD47 trended upward in LSKL-treated animals in both the LCA and RCA.

TSP-1 Is a Critical Mediator of D-Flow–Induced Stiffened Arterial Remodeling In Vivo

We used TSP-1 KO animals to test whether TSP-1 mediates d-flow–induced arterial stiffening in vivo; C57BL/6J (C57) background mice were used as the wild-type control. In both the KO and wild-type mice, the LCA developed arterial stiffening in response to d-flow (Figure 7A through 7D). But when comparing stiffness changes in the LCAs after exposure to d-flow, ex vivo biaxial testing demonstrated that TSP-1 KO mice had significantly more compliant arteries at physiological mean arterial pressures than the C57 mice (Figure 7E and 7F). These ex vivo data were then validated in vivo by using Green strain as a surrogate for arterial stiffness. Here, the LCA of C57 but not of TSP-1 KO mice demonstrated increased arterial stiffening within 3 weeks (Figure 7G).

Figure 7.

Figure 7. D-flow induced stiffening is significantly attenuated in TSP-1 KO animals.A and B, Pressure-diameter and compliance curves from ex vivo cylindrical biaxial mechanical testing of LCA and RCA from C57BL/6 (C57) animals demonstrate that the LCA is significantly stiffer than the RCA in this strain as well (n=5). C and D, Similar but less prominent differences were seen in the TSP-1 KO animals (n=7). E and F, When comparing the pressure diameter and compliance curves of the LCA (exposed to d-flow) from TSP-1 KO and C57 animals, the TSP-1 KO was significantly less stiff by pressure diameter curves beginning at 70 mm Hg and by compliance curves at 40, 60, and 70 mm Hg (n=5–7). mean±SEM; unpaired Student t test with significance (§) at P<0.0029 and P<0.0031, respectively. G, For in vivo validation, we calculated Green strain Eθθ from ultrasound imaging to identify the time course of stiffening in these animals. Here we identified increased arterial stiffness (in comparison with TSP-1 KO) in the C57BL/6 arteries exposed to d-flow to occur between weeks 2 and 3 (n=9). H, Similar to young WT arteries in vivo, the LCAs of 80-week-old animals exposed to d-flow were significantly stiffer than RCAs after 4 weeks (n=8). I, Hematoxylin and eosin staining of C57BL/6 and TSP-1 KO arteries under 4× and 20× magnification demonstrates increased myointimal hyperplasia in the LCA of C57 mice; this was not found in the TSP-1 KO mice. Elastin was visualized by autofluorescence, and we did not demonstrate differences in the elastin orientation or composition in this model. J, Graphical representation of the intima/media ratio demonstrates significant increase in myointimal hyperplasia in the C57 (but not TSP-1) arteries under d-flow (LCA) in comparison with s-flow conditions (RCA). n=7 to 8; mean±SEM; unpaired Student t test with significance set at P<0.05. Scale bar for 20× is 100 µm; scale bar for 40× is 20 µm. *P<0.05. †P<0.01. ‡P<0.001. D-flow indicates disturbed flow; KO, knockout; LCA, left carotid artery; RCA, right carotid artery; s-flow, stable flow; TGF-β, transforming growth factor-β; TSP-1, thrombospondin-1; and WT, wild-type.

After identifying that the arterial stiffening of young mouse carotid arteries by d-flow closely approximated the mechanical stiffness of unmanipulated 80-week murine carotid arteries, we next tested the impact of stiffened arteries from aging under d-flow. Here, we found that the LCA was significantly stiffer than the RCA by strain analysis by 4 weeks after partial carotid ligation (Figure 7H). Interestingly, despite an increase in collagen content from aging in the RCA of 80-week-old animals, the LCA had even greater collagen deposition at week 4 (46% dry weight in comparison with 24% in RCA). Still, there were some unique aspects of the intimal gene regulation in these 80-week-old animals that differed from those seen in young mice. TGF-β 1 and 2 were significantly downregulated in these mice, and TSP-1 and CTGF were significantly upregulated in the LCA. Col1a1, PAI1, CD36, and CD47 were not significantly different (Figure IV in the online-only Data Supplement).

Although d-flow induced myointimal hyperplasia in the LCA of C57 mice, this was not evident in the TSP-1 KO mice (Figure 7I and 7J). Similarly, the LCA of 80-week-old mice did not have an increase in myointimal hyperplasia in comparison with RCA (intimal/medial ratio of 0.09±0.04 versus 0.10±0.04; P=0.31), but they did have a significant increase in medial thickening (25±6 μm versus 22±6 μm; P=0.003).

TSP-1 Is Upregulated in Human Arteries Exposed to D-Flow

To validate these findings in patients under clinically relevant conditions, we investigated TSP-1, TGF-β, and collagen gene expression in human arteries exposed to d-flow or s-flow conditions. Validation of d-flow and s-flow conditions in human arteries was performed using ultrasound data from a representative patient. Here, we identified s-flow in the artery with unobstructed inline flow and d-flow in the arteries reconstituted distal to an arterial occlusion. The hemodynamic environment in the superficial femoral artery was characterized by unidirectional flow throughout the cardiac cycle (ie, nonreversing) and a time-averaged WSS value of 23.4±17.6 dyne/cm2 (Figure 8A). Conversely, the hemodynamic environment distal to occlusion in the posterior tibial artery was observed to be complex with oscillatory WSS from adjacent retrograde (Figure 8B) and antegrade (Figure 8C) flow patterns. D-flow conditions were further supported by time-averaged WSS values in the posterior tibial artery exhibiting low WSS in comparison with the superficial femoral artery with inline flow (9.5±4.7 and 15.6±4.7 dyne/cm2, respectively) (Figure 8D).

Figure 8.

Figure 8. TSP-1 and collagen genes are increased in human arteries exposed to d-flow.A through D, Human peripheral arteries with inline (nonobstructive) flow have s-flow, whereas those arteries distal to obstruction have d-flow. A, Inline flow is demonstrated in a representative patient’s superficial femoral artery on arterial duplex. B and C, Distal to the occlusion there is flow reversal, which is manifest as retrograde mixed with antegrade filling of the posterior tibial artery and consistent with oscillatory WSS. D, Modeling of WSS in these images during antegrade flow demonstrates low WSS in the posterior tibial artery (distal to occlusion) in comparison with normal WSS in the superficial femoral artery with inline flow, representing s-flow. E, TSP-1 is significantly increased in human arteries under d-flow conditions in comparison with s-flow. n=4 for s-flow human arteries, n=6 for d-flow human arteries; mean±SEM; unpaired Student t test with significance set at P<0.05. F, IHC demonstrates increased TSP-1 expression in d-flow regions of human arteries in comparison with s-flow regions (L: lumen of artery; scale bar is 500 μm). G through I, Col1a1 is increased under d-flow in our murine model in the endothelium and the media/adventitia of the LCA in comparison with RCA (s-flow). Col4a1 and Col16a were not significantly affected by d-flow in our murine model. n=3; mean±SEM; paired t test with significance set at P<0.05. J through L, In human arteries, both Col1a1 and Col4a1 are upregulated under d-flow in comparison with s-flow conditions. Col16a1 is not altered by d-flow human arteries. n=4 for s-flow human arteries, n=6 for d-flow human arteries; mean±SEM; unpaired Student t test with significance set at P<0.05. *P<0.05. †P<0.01. P‡<0.001. DAPI indicates 4′,6-diamidino-2-phenylindole; d-flow, disturbed flow; EC, endothelial cell; IHC, immunohistochemical; LCA, left carotid artery; RCA, right carotid artery; s-flow, stable flow; TSP-1, thrombospondin-1; and WSS, wall shear stress.

Next, we compared TSP-1 expression in human arteries exposed to d-flow or s-flow conditions and found a significant increase in TSP-1 expression in human arteries exposed to d-flow in comparison with s-flow conditions (Figure 8E). Immunohistochemistry confirmed increased TSP-1 expression in the arteries exposed to d-flow (Figure 8F). There was also a significant increase in Col1a1 expression in the d-flow human arteries, similar to that seen in the murine arteries (Figure 8G and 8J, *P<0.05). In alignment with our murine model, human arteries exposed to d-flow also had elevations in Col4a1 (Figure 8H and 8K, *P<0.05) and TGF-β1 expression (Figure V in the online-only Data Supplement), and decreased expression of TGFβR3. Table I in the online-only Data Supplement lists the patient sex, ages, and comorbidities from which the human arteries were collected.

Discussion

Flow-Mediated Murine Model of Stiffened Arterial Remodeling

It is exciting that this model demonstrates that d-flow induced arterial stiffening through collagen deposition after partial carotid ligation in 12-week-old mice that is mechanically similar to that of unmanipulated 80-week-old mice. In an a priori fashion, we used pathway analysis of intimal gene expression in this model to identify a critical role for TGF-β signaling pathways. We discovered that TSP-1 stimulated profibrotic genes to promote stiffening. We then validated these findings in human arteries exposed to d-flow. To our knowledge, this is the first report identifying a critical role for TSP-1 in the mechanical stiffening of arteries and the localization of TSP-1 to areas of d-flow in human arteries of patients with PAD.

TSP-1 has previously been linked to PAD, but the mechanism of upregulation is not known. Given the direct relationship of arterial stiffness and PAD with aging, our validation of TSP-1 in human arteries under d-flow may explain in part why TSP-1 is upregulated in patients with PAD.30,31 TSP-1 can also influence vascular tone and nitric oxide signaling.13,19 Specifically, it has been reported that TSP-1 KO animals have improved vasodilation through CD4732 that may be protective of the adverse effects of d-flow on EC dysfunction. In our model, neither CD36 nor CD47 gene expression was significantly affected by flow conditions in the carotid endothelium and did not appear important to our model. Still, it is highly likely that cross talk does exist in arteries between CD36 and CD47 and TSP-1/TGF-β pathways as has been recently reported in pericytes.33 Finally, it is likely that there are redundant pathways that affect vascular tone and EC dysfunction that may also contribute to arterial stiffening,1 but the focus of this work was to identify the molecular mediators of d-flow–induced mechanical stiffening (as opposed to vascular tone).

TSP-1 Pathways in Fibrosis and Arterial Remodeling

TSP-1 is a large (450 kDa) matricellular protein that is found in the intima and media of atherosclerotic arteries and is important to arterial remodeling attributable to injury or hypertension.30,3436 TSP-1 can promote fibrosis in wound healing through both TGF-β–dependent and –independent mechanisms37. In the TGF-β–dependent pathway, TSP-1 activates latent TGF-β through its LAF.38,39 Active TGF-β then binds sequentially to TGFβRs that promote downstream cascades regulating the extracellular matrix.40,41 It is exciting that these pathways have a number of pharmacological targets including blocking the activation of TGFβ by TSP-1 (LSKL) and TGFβR/Smad protein inhibitors.42,43 TSP-1 can also promote fibrosis in TGF-β–independent pathways by binding to calreticulin in an Akt-dependent manner,44 and matrix metalloproteinases are activated by TSP-1 through its binding with low-density lipoprotein receptor–related protein.45 Here, we demonstrate significant attenuation of arterial stiffening in TSP-1 KO mice and global downregulation of profibrotic genes in LSKL-treated mice. However, the precise contribution of these TSP-1 pathways in d-flow–induced arterial stiffening remains to be discovered.

Arterial stiffening is closely linked to aging, and TGF-β1 has been found to promote adventitial collagen I and III in aged mice.6 This is similar to our findings of collagen deposition in response to d-flow in young (12- to 20-week-old) mice. Thus, the rapid onset in this model may be instructive to mechanisms of aging. It is important to note that arterial stiffening may also be a modifiable intermediary condition affecting atherosclerotic plaque formation and progression. Unfortunately, to date, the role of stiffness in atherosclerotic arterial remodeling is unclear from the literature. Gotschy et al46 identified by MRI that local arterial stiffness preceded atherosclerotic plaque formation in the ApoE–/– mouse model of atherosclerosis. Van Herck et al47 modified ApoE–/– KO mice to disrupt elastin formation and found that atherosclerosis formed in stiffened arteries and that more arterial stiffness was associated with more atherosclerotic plaque. However, recently, the direct relationship between stiffness and atherosclerosis has been called into question by the Wagensiel group using a combination of elastin-deficient and Ldlr KO mice.48 Prior work in the Jo laboratory has demonstrated that combining partial carotid ligation with a high-fat diet in ApoE–/– mice led to rapid atherosclerotic plaque formation in d-flow regions,24 and given the model and our findings in this article, it is highly likely that stiffening preceded these plaques. It is interesting to note that, in a double-knockout (TSP-1–/–/apoE–/–) mouse model, Moura et al49 found a mixed effect of TSP-1 on atherosclerosis in the aortic root of mice over time. They had a delay to peak plaque volume with no difference in total late plaque volume, but these animals did have greater inflammation and necrotic core formation in their atherosclerotic plaques. It is unclear what effect would be found if we added an atherogenic environment into the TSP-1 KO’s remodeling to d-flow.

Our novel discovery of TSP-1 mediating d-flow–induced arterial stiffening may help reconcile these discrepancies in the literature. Specifically, we propose a role for TSP-1 (under d-flow conditions) in the amplification of inflammatory signaling pathways that contribute both to arterial stiffening and possibly the onset and progression of focal atherosclerotic plaque formation (Figure VI in the online-only Data Supplement). The modifiability of this pathway seen in the LSKL treatments and TSP-1 KO supports these pathways having promise for PAD therapies and suggests that such therapies may improve cardiovascular health in d-flow arterial regions broadly. Here, we used normal S129 and C57Bl/6J mice that did not have their lipids molecularly manipulated in our d-flow model of arterial stiffness. These mice did not develop atherosclerosis, but they did develop myointimal hyperplasia, which is thought to contribute to atherosclerosis.12 It is interesting to note that myointimal hyperplasia was not present in the LCA of TSP-1 KO mice, and these arteries were also relatively protected from stiffening under d-flow conditions. Because TGF-β has been implicated in the development of myointimal hyperplasia,50 it is not clear if the lack of intimal hyperplasia in TSP-1 KO arteries exposed to d-flow is attributable to decreased stiffness, or if this too is caused by TSP-1–mediated activation of TGF-β. Moura et al34 have previously published that the TSP-1 KO animals undergoing complete (not partial) carotid ligation can mount a myointimal response that is related to smooth muscle cell activation. However, even in this model, the absolute myointimal response was limited in the TSP-1 KO animals, which is consistent with our findings in this partial carotid ligation model. Further support of our findings has been demonstrated with TSP-1 antibodies decreasing neointima in a denuded carotid artery model of EC injury–driven arterial remodeling.51

Limitations

Still, there are limitations to our study that deserve discussion. First, this model has been focused on the EC response to d-flow, but there are known strain forces that are sensed by the arterial media explaining the transmural upregulation of TSP-1 seen in Figure 4. Although it is not surprising that stiffened arterial remodeling is a transmural process, it is notable that in vivo and in vitro testing performed here support the hypothesis that the EC response to d-flow is critical to this process. In addition, the association of TSP-1 and collagen gene upregulation in human arteries exposed to d-flow supports the clinical relevance of this pathway. Second, although the mechanical analyses of this model demonstrate similar stiffness in d-flow–exposed LCA from young mice with unmanipulated carotid arteries in 80-week-old mice, our d-flow model may not exactly mimic the arterial stiffening manifest by hypertension and aging.52 This may account for the slight differences in the EC response to d-flow between the younger and older mice. However, the upregulation of TSP-1, CTGF, and increased collagen deposition remained very similar. It is also important to recognize that the human arteries that were exposed to d-flow necessarily came from patients with PAD who had blockage proximal to the arteries interrogated. The human arteries exposed to s-flow came from patients of similar ages who did not have obstructive PAD. The ideal comparison of arteries would have been to examine arteries from the same patient under s-flow and d-flow conditions. However, because it is rare to perform a major amputation above the level required for healing by the vascular supply, comparable human arteries under both s-flow and d-flow conditions are rare, so this was not feasible in this study. Here, larger animal models of d-flow such as our porcine model of PAD could be used to definitively determine in a large animal the direct link between d-flow and TSP-1.53 Finally, hypertension certainly promotes medial thickening and pathological vascular smooth muscle cell behavior, and its absence in our model (Table II in the online-only Data Supplement) may underestimate the role of the media (and vascular smooth muscle cells) in stiffened arterial remodeling in hypertensive populations. Nevertheless, this model’s rapid and progressive arterial stiffening with collagen deposition (mimicking aging) may be very useful in determining the role of more and less stiff arteries in the development of focal atherosclerotic plaque formation. Specifically, the relative lack of stiffening under d-flow in TSP-1 KO mice uniquely enables this model to be modified with hypertensive or atherogenic conditions to answer clinically important questions in PAD.

Conclusions

We have developed a flow-dependent in vivo murine model of arterial stiffness that is mechanically similar to that of aged mice and identified a critical role for TSP-1 in d-flow–induced arterial stiffening. These findings were validated in arteries from d-flow regions of patients with PAD. This model of the TSP-1 pathways involved in arterial stiffness holds promise for the development of novel therapies that can reverse this arterial pathology before atherosclerotic plaque forms and irreversible damage has occurred. Such therapies may be helpful in promoting arterial health during aging and in patients with cardiovascular disease.

Acknowledgments

Part of this work was recognized with the Jay D. Coffman Early Career Investigator Award by the Peripheral Vascular Disease council of the American Heart Association and presented as an oral presentation at the 2014 American Heart Association’s Scientific Sessions in Chicago, IL. The authors would also like to thank Sebastian Perez from Emory University’s Department of Surgery for his assistance with the statistical analysis of this work.

Footnotes

*Drs Kim, Pokutta-Paskaleva, and Kumar contributed equally.

The online-only Data Supplement is available with this article at http://circ.ahajournals.org/lookup/suppl/doi:10.1161/CIRCULATIONAHA.116.026361/-/DC1.

Circulation is available at http://circ.ahajournals.org.

Correspondence to: Luke P. Brewster, MD, PhD, Assistant Professor of Surgery, Division of Vascular Surgery, Emory University; Staff Surgeon, Atlanta VA Medical Center; Surgical and Research Services, Emory University School of Medicine, 101 Woodruff Cir, WMB Suite 5105, Atlanta, GA 30322, or Hanjoong Jo, PhD, Coulter Department of Biomedical Engineering, Emory University and Georgia Institute of Technology, 1760 Haygood Dr, E170 HSRB, Atlanta, GA 30306. E-mail , or

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