This article has Open Peer Review reports available.
Fluid upstream shear stress of rabbit aortic stenosis inhibits neointimal hyperplasia by promoting endothelization after balloon injury
- Jinxue Liu†1,
- Yucheng Peng†1,
- Junxing Lai1,
- Weidong Gao1,
- Anjian Song1 and
- Gaoxing Zhang1Email authorView ORCID ID profile
© The Author(s). 2017
Received: 23 May 2017
Accepted: 2 October 2017
Published: 30 October 2017
Atherosclerosis is associated with disturbed blood flow characterized by low and oscillatory shear stress (SS), however, few study directly links SS to neointimal hyperplasia in animal model. This study was focused on the effects of changed SS upon the neointimal hyperplasia which responded to balloon injury in a novel rabbit model with partially-constricted abdominal aorta.
We established a rabbit model subjected to partial abdominal aortic constriction with a cylinder-shaped cannula as a model of disturbed flow, which was similar to the hemodynamic features of stenosis caused by atherosclerosis plaque. Further, balloon injury was performed to investigate the relationship between SS and neointimal hyperplasia. Four weeks later, the abdominal aorta was assessed with digital subtraction angiography (DSA) and intravascular ultrasound (IVUS). The vascular sections were embedded in paraffin blocks for morphometric analysis to evaluate neointimal hyperplasia, and anti-CD31 immunohistochemical staining was for endothelialization ratio.
In upstream the stenosis, the changed SS leads to neointimal hyperplasia compared with normal SS (11,729 ± 1205 vs 8418 ± 737, P = 0.023). However, the upstream SS of the stenosis can promote vascular re-endothelialization after balloon injury compared with normal SS, verified by endothelialization ratio (0.36 ± 0.03 vs 0.32 ± 0.03, P = 0.017), thereby attenuate neointimal hyperplasia (64,851 ± 3995 vs 68,335 ± 3867, P = 0.018).
The upstream SS of stenosis, not downstream SS, inhibits the neointimal hyperplasia after balloon injury by promoting vascular re-endothelializtion.
Atherosclerosis and related complications are the leading cause of mortality in most countries. Though a lot of systemic factors, including such as dysglycemia, hypercholesterolemia, hypertension and smoking, are identified as risk factors, disturbed flow was more likely to play a pivotal role in atherosclerosis development with a nonrandom but preferentially pattern, which was characterized by low and oscillatory wall shear stress (SS) in branched or curved arteries [1, 2]. Increasing evidences have indicated that SS was the chief culprit in the development of vascular injury. With the applications of intravascular ultrasound (IVUS) and computational fluid dynamics, researchers demonstrated that SS could be used to predict in-stent restenosis-induced neointimal formation and assess post-balloon angioplasty vascular remodeling progress . Research has shown that high SS is associated with in-stent restenosis and target lesion revascularization, even in successful balloon angioplasty cases . However, the relationship between SS and neointimal hyperplasia induced by balloon angioplasty needs to be further confirmed.
Vascular endothelial cells (ECs) are the essential parts of the inner lining inblood vessel wall and always directly exposed to disturbed blood flow. Meanwhile, ECs could be influenced by various chemical and mechanical stimuli, which finally regulated homeostatic functions . EC dysfunction is a vital pathology factor in vascular diseases, including atherosclerosis and thrombosis. In recent years, a variety of models in vitro have been established to regulate shear flow to ECs, which are aimed to mimic important features of ECs flow environments in vivo. These models helped researchers to explore detailed mechanisms of their responses to SS, including the effects on EC morphology, cytoskeletal organization, proliferation, migration, permeability and junctional proteins, EC signaling and gene expression [1, 4, 5].
Multiple studies have explored effect of SS on vascular structure in vitro [4, 5]. However, the role of SS in the vascular remolding and neointimal formation has not been completely clarified, especially disturbed flow in vivo. In addition, an appropriate model should be established to explore this issue. Thus, it is important for better clinical understanding to establish an animal model in which disturbed flow can be acutely induced. On the other hand, previous study indicated rabbits were ideal animals for vascular model . Accordingly, we enrolled the rabbits subject to transverse abdominal aortic constriction with a cylinder-shaped cannula, which mimics important hemodynamic features of stenosis caused by atherosclerosis, to explore the generated SS on the effect of the vascular remolding after balloon injury in vivo. We hypothesized that this constrictive vascular model, similar to the atherosclerotic plaque, will cause varying degrees of intimal hyperplasia in the upstream and downstream of the constricted vessel, with or without balloon injury.
All experimental protocols complied with the guidelines for the Chinese Animal Care and Use Committee standards. All in vivo procedures were also performed in accordance with protocols approved by the Animal Ethics Committee of Sun Yat-sen University. 40 New Zealand white rabbits were purchased from cavenstech company (Changzhou, China, permission code for experimental animals: SCXK2016–0010), weighing 2.2–2.6 kg, male, were randomly divided into 5 groups (n = 8 per group), were housed individually in steel mesh cages and fed normal rabbit chow and water for at least 1 week prior to operative procedures. The rabbit was anesthetized with 3% pentobarbital (1 ml/kg) and then the right femoral artery was cannulated with a four introducer sheath. All catheters were subsequently introduced through this sheath and advanced to the abdominal aorta through a 0.014-in. guidewire. Heparin sodium (200 IU/kg) was intra-arterially injected. After that, a midsagittal incision was made and the middle vessel of the renal artery to the common iliac artery about 1 cm in length was separated, waiting for the next step which is decided by the grouping. Baseline angiogram and IVUS of the abdominal aorta were obtained for each rabbit. All operations were under the sterile condition.
Four weeks later, the rabbit was checked with DSA and IVUS again and then euthanized with an overdose of pentobarbital, the aorta was dissected from the renal artery to iliac bifurcation was perfusion-fixed with 10% neutral formaldehyde overnight. The fixed aorta was embedded in paraffin blocks for morphometric and immunohistochemical studies.
Morphometric analysis was performed with the Image-Pro Plus 6.0 image analyze system by an independent observer blinded to the study groups. Cross-sections were obtained from two different sites, the very upstream and downstream sections of the constricted artery or sham groups. The tissue samples were cut into serial 5 μm cross-sections for general histological staining with hematoxylin and eosin (HE) and Verhoeff-Van Gieson. Image-Pro Plus 6.0 image analyze system was performed to determine lumen area (LA, pixel) and internal elastic lamina area (IELA, pixel), and neointima area (NA, pixel) was calculated using the following equation: NA = IELA-LA. The mean area in the corresponding segment (n = 3 sections per segment) was used for statistical analysis.
Immunohistochemistry for paraffin embedded cross sections was described previously . In brief, after deparaffinization and hydration, sections were treated with 3% H2O2 in PBS for 10 min to inhibit intrinsic peroxidase activity and 5% blocking solution for 20 min to prevent non-specific antibody binding. Antigen retrieval was performed for cross sections with citrate buffer (pH 6.0). They were then incubated overnight at 4°Cwith CD31 monoclonal antibody (1:50; Abnova). After rinsing in PBS, slides were then incubated for 20 min at room temperature with goat anti-rat IgG, followed by 20 min for SABC complex. Slides were rinsed again with PBS and were stained with DAB, counterstained in hematoxylin, dehydrated, and mounted. Control immunostaining was carried out by the same procedure in which the primary antibody was replaced by nonimmunized serum.
The results of HE staining and immunohistochemical staining were examined by a professional pathological doctor using the Image-Pro Plus 6.0 image analyze system . Five views on every slides were chosen randomly and the area density of positive expression (positive area/total area) was quantified. Results are described as mean ± SD. Statistical tests were performed with SPSS (version 13.0; SPSS Inc., Chicago, Ill), using Student t test for 2 groups or one-way ANOVA followed by multiple comparisons with Bonferroni test. Probability values of P < 0.05 was considered as significant.
Establishment of Rabbit model
Four rabbits (two in group 2, one in each of the group 3 and group 4) died of excessive anesthesia, thrombosis, hemorrhagic shock, balloon injury during the experiment and the remaining rabbits completed the entire study. The average weight of rabbits 2.36 ± 0.25 kg at baseline but increase to 3.14 ± 0.16 kg four weeks later. There was no significant difference in body weight between the five groups (dates not shown), suggesting model was successfully established. Hemodynamic assessments were performed to confirm changing of vascular SS after partial abdominal aortic constriction. These routine measurements are assessed to confirm the model of changed SS.
The upstream SS of stenosis attenuates the lumen loss after balloon injury
The upstream SS of stenosis induces neointimal hyperplasia in the normal vessel but alleviates the hyperplasia after balloon injury
The upstream SS of stenosis accelerated re-endothelialization after balloon injury, but in contrary to the effects of downstream SS
SS is a biomechanical force generated by fluid flow on the surface of the endothelium and involved in all stages of vascular physiology or diseases . However, the effect of disturbed SS, induced by vascular constriction after the balloon injury, on the neointimal hyperplasia has not been fully understood in vivo. Cheng et al.  has established a perivascular SS model in mouse carotid arteries which exposed vascular upstream regions under influence of low and unidirectional SS, while downstream under low and oscillatory (ie, with vortices) SS, and they explore the plaque formation and composition with these models.
The primary sensors of mechanical forces in the vessel wall are endothelial cells. A considerable amount of literature on EC responses to SS has documented about EC morphology , function , and gene expression , and on the differentiation of immature cells, such as endothelial progenitor cells (EPCs) and embryonic stem (ES) cells . As a primary enzyme that produces nitric oxide (NO), endothelial nitric oxide synthase (eNOS) mainly regulated blood vessel relaxation and its expression could be influenced by various mechanical forces, including SS [7, 11]. Re-endothelialization is beneficial to suppress neointimal hyperplasia with vascular endothelial growth factor (VEGF) coated stents after balloon injury . In our study, we demonstrated that the upstream SS undermines the integrity of the endothelial cells, which further impact the function of endothelial cell and vascular homeostasis, thus lead to the neointimal hyperplasia in normal vessel. Moreover, the proximal SS inhibited the neointimal hyperplasia after balloon injury by means of accelerating re-endothelialization of the injured artery, but this effect was not maintained in the downstream SS.
Previous study showed that disturbed flow of a step-flow channel damaged ECs more severely than blood flow in static conditions . In contrast, a high level of laminar SS inhibited proliferation of ECs  via reducing DNA synthesis . Thus, the models of low SS leads to early neointimal hyperplasia probably via more complicated mechanisms, including enhanced SMC migration or proliferation, upregulation of multiple factors (VCAM-1, PDGFs, MMP-9), aberrant monocyte adhesion and uncontrolled macrophage infiltration . Restriction of the common carotid artery (CCA) by ligating the distal CCA was performed in previous study to mimic low SS, and low SS in this model significantly increased the neointimal thickness after 4 weeks CCA ligation .This point was also demonstrated in our study that on the upstream and downstream of stenosis, neointimal hyperplasia was found at the low laminar SS district in the normal vessel. Accordingly, our animal model, by narrowing the vessel instead of reducing blood flow, can more accurately mimic hemodynamic changes caused by artery stenosis, such as atherosclerosis plaque in clinical scenario.
The structural integrity and functional completeness of the endothelial monolayer directly influenced vascular homeostasis. As a common physiological process in multiple vascular diseases, vascular remodeling responds to long-term effect of disturbed hemodynamic conditions, and further contributes to the pathophysiology progress of intima-media thickening (IMT), which accelerated progress of vascular diseases . The effect of SS upon endothelium is a well-kown determinant of vascular remodeling. For instance, vessel diameter was abnormally increased by disturbed blood flow acceleration attributable to an arteriovenous shunt. Conversely, blood flow reduction results in the cascade reaction of signaling pathways which lead to atrophy of target vascular lumen . In our study, the downstream low, oscillatory (ie, with vortices) SS leads to outward remolding with predominantly increased lumen and attenuation in the external elastic lamina, the reason may be the changing SS to achieve a homeostasis balance.
Although there has been evidence that low SS was involved in the pathophysiologic process of restenosis , but there are also reports that successful balloon angioplasty induced high SS which contributed to restenosis and target lesion revascularization, or during the follow-up period . These seemingly contradictory conclusions illustrate the complexity of SS on the role of neointimal hyperplasia . Chow et al. showed that pathological levels of SS (>3 N/m2) initiated platelet aggregation . This view also can be used to explain our finding in the upstream of stenosis, in which low unidirectional SS inhibits the activation, deposition of platelet and weaken the neointimal hyperplasia ultimately.
Appropriate low unidirectional SS is beneficial for the inhibition of intimal hyperplasia after balloon angioplasty, and it is a potential management target of in-stent stenosis.
We gratefully acknowledge the support of pathology department in our hospital.
Availability of data and materials
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.
JXLi: Study concepts, Study design. YCP: Study concepts, Study design. JX La: Literature research, Clinical studies, Experimental studies. WDG: Data acquisition, Data analysis, Statistical analysis. AJS: Data acquisition, Data analysis, Statistical analysis. GXZ: Clinical studies, Experimental studies, Guarantor of integrity of the entire study. All authors have read and understand manuscript; all authors approved the content of the study in manuscript to be published.
Ethics approval and consent to participate
All in vivo procedures were also performed in accordance with protocols approved by the Animal Ethics Committee of Sun Yat-sen University.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Chiu JJ, Chien S. Effects of disturbed flow on vascular endothelium: pathophysiological basis and clinical perspectives. Physiol Rev. 2011;91(1):327–87.View ArticlePubMedGoogle Scholar
- Santos-Gallego CG, Picatoste B, Badimón JJ. Pathophysiology of acute coronary syndrome. Curr Atheroscler Rep. 2014;16(4):401.View ArticlePubMedGoogle Scholar
- Wentzel JJ, Corti R, Fayad ZA, Wisdom P, Macaluso F, Winkelman MO, Fuster V, Badimon JJ. Does shear stress modulate both plaque progression and regression in the thoracic aorta? Human study using serial magnetic resonance imaging. J Am Coll Cardiol. 2005;45(6):846–54.View ArticlePubMedGoogle Scholar
- Thury A, van Langenhove G, Carlier SG, Albertal M, Kozuma K, Regar E, Sianos G, Wentzel JJ, Krams R, Slager CJ, Piek JJ, Serruys PW, DEBATE Investigators. Doppler Endpoints Balloon Angioplasty Trial Europe. High shear stress after successful balloon angioplasty is associated with restenosis and target lesion revascularization. Am Heart J. 2002;144(1):136–43.View ArticlePubMedGoogle Scholar
- Chien S. Mechanotransduction and endothelial cell homeostasis: the wisdom of the cell. Am J Physiol Heart Circ Physiol. 2007;292(3):H1209–24.View ArticlePubMedGoogle Scholar
- Chen Z, Tzima E. PECAM-1 is necessary for flow-induced vascular remodeling. Arterioscler Thromb Vasc Biol. 2009;29(7):1067–73.View ArticlePubMedPubMed CentralGoogle Scholar
- Cheng C, Tempel D, van Haperen R, van der Baan A, Grosveld F, Daemen MJ, Krams R, de Crom R. Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress. Circulation. 2006;113(23):2744–53.View ArticlePubMedGoogle Scholar
- Cheng C, van Haperen R, de Waard M, van Damme LC, Tempel D, Hanemaaijer L, van Cappellen WGA, Bos J, Slager JC, Duncker DJ, de van Steen WFA, de Crom R, Krams R, et al. Shear stress affects the intracellular distribution of eNOS: direct demonstration by a novel in vivo technique. Blood. 2005;106(12):3691–8.View ArticlePubMedGoogle Scholar
- Helmke BP, Goldman RD, Davies PF. Rapid displacement of vimentin intermediate filaments in living endothelial cells exposed to flow. Circ Res. 2000;86(7):745–52.View ArticlePubMedGoogle Scholar
- Ando J, Yamamoto K. Vascular mechanobiology: endothelial cell responses to fluid shear stress. Circ J. 2009;73(11):1983–92.View ArticlePubMedGoogle Scholar
- Kolluru GK, Sinha S, Majumder S, Muley A, Siamwala JH, Gupta R, Chatterjee S. Shear stress promotes nitric oxide production in endothelial cells by sub-cellular delocalization of eNOS: A basis for shear stress mediated angiogenesis. Nitric Oxide. 2010;22(4):304–15.View ArticlePubMedGoogle Scholar
- Tang C, Wang G, Wu X, Li Z, Shen Y, Lee JC, Yu Q. The impact of vascular endothelial growth factor-transfected human endothelial cells on endothelialization and restenosis of stainless steel stents. J Vasc Surg. 2011;53(2):461–71.View ArticlePubMedGoogle Scholar
- Li YS, Haga JH, Chien S. Molecular basis of the effects of shear stress on vascular endothelial cells. J Biomech. 2005;38(10):1949–71.View ArticlePubMedGoogle Scholar
- Akimoto S, Mitsumata M, Sasaguri T, Yoshida Y. Laminar shear stress inhibits vascular endothelial cell proliferation by inducing cyclin-dependent kinase inhibitor p21(Sdi1/Cip1/Waf1). Circ Res. 2000;86(2):185–90.View ArticlePubMedGoogle Scholar
- Koskinas KC, Chatzizisis YS, Antoniadis AP, Giannoglou GD. Role of endothelial shear stress in stent restenosis and thrombosis: pathophysiologic mechanisms and implications for clinical translation. J Am Coll Cardiol. 2012;59(15):1337–49.View ArticlePubMedGoogle Scholar
- Sullivan CJ, Hoying JB. Flow-dependent remodeling in the carotid artery of fibroblast growth factor-2 knockout mice. Arterioscler Thromb Vasc Biol. 2002;22(7):1100–5.View ArticlePubMedGoogle Scholar
- Korshunov VA, Berk BC. Flow-induced vascular remodeling in the mouse: a model for carotid intima-media thickening. Arterioscler Thromb Vasc Biol. 2003;23(12):2185–91.View ArticlePubMedGoogle Scholar
- Papafaklis MI, Bourantas CV, Theodorakis PE, Katsouras CS, Naka KK, Fotiadis DI, Michalis LK. The effect of shear stress on neointimal response following sirolimus- and paclitaxel-eluting stent implantation compared with bare-metal stents in humans. JACC Cardiovasc Interv. 2010;3(11):1181–9.View ArticlePubMedGoogle Scholar
- Kawano K, Yoshino H, Aoki N, Udagawa H, Watanuki A, Hioki Y, Hasumura Y, Yasumura T, Homori M, Murata M, Ikeda Y, Ishikawa K. Shear-induced platelet aggregation increases in patients with proximal and severe coronary artery stenosis. Clin Cardiol. 2002;25(4):154–60.View ArticlePubMedGoogle Scholar
- Chow TW, Hellums JD, Moake JL, Kroll MH. Shear stress-induced von Willebrand factor binding to platelet glycoprotein Ib initiates calcium influx associated with aggregation. Blood. 1992;80(1):113–20.PubMedGoogle Scholar