Endovascular interventions are inevitably linked to the vessel wall injury and associated healing response, which is initiated as the physiological reaction of the artery aimed to replenish defects in the tissue. Excessive vascular healing and related complications may be a direct cause for the necessity of repeated surgical interventions, as well as increased morbidity and mortality. Understanding the molecular biology underlying the reparative capacity of the vascular wall may facilitate the development of novel approaches and pharmacological treatments available to patients to minimize the risk of post-surgery adverse events.
3.3.1 Animal strains used
As in vitro cell culture cannot recapitulate the complexity of living organisms with intricate networks of interconnected and interdependent cells, tissues and organs, several in vivo animal models have been utilized for the purpose of investigating molecular mechanisms of SMC phenotypic modulation.
Sprague-Dawley wild-type rats are commercially available, best-studied rat strain in experimental research of vascular wall healing. Due to the ease of handling, calmness and high survival rates, they have been a top choice in both academic and industry-driven in vivo studies. This particular strain was utilized to generate results in Studies I-IV and its selection was dictated predominantly by the previous experience of our lab with these animals, in-house expertise with balloon injury model, as well as availability and possibility to compare obtained results with published literature.
Generation of Pcsk6-/- mice was previously comprehensively described in the publication from Daniel B. Constam and Elizabeth J. Robertson (Harvard University, Cambridge, Massachusetts, USA)386 and these mice were generously provided by Prof Anne-Marie Malfait, Rush University, USA. Briefly, using Pcsk6 gene targeting vector designed to delete approximately 280 bp from the coding exon region, the successful loss-of-function mutation was obtained, which resulted in the generation of Pcsk6-/- mice with globally abolished Pcsk6 expression. These were further backcrossed to C57BL/6J background for at least ten generations in order to remove the genetic variation from the original strains. Importantly, homozygous mutants display 25% embryonic lethality, associated predominantly with cardiac developmental defects386, 387. No obvious cardiac defects have been observed in the surviving offspring388. Phenotypically, Pcsk6-/- mice are of chinchilla color, have no pigment in the iris and some of them display complex craniofacial malformations including cyclopia.
In Study II we utilized male, age-matched (4-6 months old) C57BL/6J control animals and viable Pcsk6-/- mice. The use of male sex was justified by the fact that females were reported to display phenotypic changes in the reproductive system, including ovarian pathology, likely linked to hormonal disbalance387.
Until recently, it was impossible to track SMCs in vivo, as during phenotypic modulation these cells lose their classical contractility markers and acquire phenotypic and functional features of a multitude of other cell types. In order to reliably identify SMC-derived cells within mouse atherosclerotic lesions and confirm in vivo BCL2 Associated Transcription Factor 1 (BCLAF1) involvement in processes related to survival/apoptosis and SMC transdifferentiation into macrophage-like cells, in Study III we utilized fixed brachiocephalic arteries from Tomato Myh11 lineage-tracing mice generously provided by Prof Nicholas Leeper (Stanford University, Stanford, CA, USA). Animals were generated, as previously described389, by crossing female B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze /J
(007914, Jackson Laboratory) mice with male Myh11-CreERT2, ApoE-/- ones. Cre recombinase was activated by repeated intraperitoneal tamoxifen injections taking place for 5 days from when the mice reached 8 weeks of age. Then mice were fed a high-fat Western Diet for 18 weeks in order to accelerate atherosclerotic lesion formation and brachiocephalic arteries were collected for further studies. Importantly, utilization of the male lineage-tracing mice for the purpose of immunofluorescent co-localization stainings in this study was imposed by the Cre allele being initially inserted into the Y chromosome.
3.3.2 Rat carotid artery balloon injury model
Rat carotid balloon injury is one of the best characterized and most frequently used animal models to investigate vessel wall healing with intimal hyperplasia formation and associated SMC phenotypic modulation. Briefly, male Sprague-Dawley rats (300-400g) under isoflurane-induced anesthesia were subjected to an arteriotomy, where a 2-F Fogarty catheter was inserted into the left common carotid artery. By repeated movements of the inflated catheter along the aorta with simultaneous rotation, the mechanical injury associated with denudation of the protective endothelial layer was inflicted on the arterial wall, which subsequently resulted in the initiation of a healing response. As comprehensively shown before by us and others11, 390, 391, removal of the endothelium and direct exposure of medial SMCs to the blood flow, triggers a cascade of complex events, which according to global transcriptomic profiling and confirmatory immunostainings, can be divided into three phases:
early, intermediate and late response (Figure 10). The acute phase, which encompasses 2-10h timepoints, has been predominantly associated with enrichment of processes such as coagulation and inflammation with immediate early induction of various cytokines and chemokines (Ccl2, Cxcl1, or Clec1b). Moreover, as expected, it is also prominently marked by the involvement of transcription factors Sp1 and Smad3 regulating phenotypic modulation of SMCs, as well as Tp53 and Brca1 related to apoptosis of SMCs11, which when sustained for up to 24h may lead even up to 70% reduction in the vessel wall cellularity167. Intermediate response stretches from 20h up to 5 days and while there is still no evidence of neointima formation, remaining SMCs are stimulated with various molecules from the blood and adherent platelets, such as Pdgfb, which results in their major activation, characterized by extensive loss of myofilaments and repression of the cytoskeleton related proteins (Smtn, Acta2, Myh11 and Tagln)11. At the same time, enlargement and expansion of the endoplasmic reticulum (ER) and Golgi complex can be seen in these cells60. Concomitantly, upregulation of markers related to ECM remodeling (Tnn1, Thbs4), as well as cell proliferation (Cdkn3) and migration (matrix metalloproteinase 12) was observed. Interestingly, this phase was also marked by the upregulation of CD34, as well as transcription factors Pou5fl (Oct4) and Pax311, all likely related to clonal expansion of SMCs. This seems to be in accordance with recent reports showing that response to injury is associated with phenotypic modulation of SMCs within the media, but neointima arises mostly due to the extensive proliferation of a small subset of oligoclonal SMCs242. Fourteen days after endothelial denudation a prominent neointima is formed, and it consists of multiple layers of phenotypically modulated SMCs and accumulated collagen- and elastin-rich ECM. Already at this point, SMCs closer to the media show signs of partially restored contractile phenotype, namely a reduction in the number of secretory organelles with an accompanying increase in the myofilaments60, 61. The late phase of the healing response taking place between 2-12 weeks is dominated by attenuation of SMC proliferation and reinstatement of contractile machinery, signifying successful restoration of vessel wall integrity11.
As arteries consist predominantly of SMCs and these cells are the major responders to vascular wall injury, this model is excellent for studying their phenotypic modulation. Single-cell sequencing and associated deconvolution analyses elevated the relevance of research
findings related to this model, as by enabling the identification of cell-specific molecular mechanisms on an unprecedented level, they consequently identified SMC conversion from contractile to synthetic phenotype to be the dominant characteristic of the healing vascular wall. In Studies I – IV, we utilized the BiRCA in-house generated resource (acronym Biobank of Rat Carotid Artery), where a systematic biobanking approach was applied to the rat carotid balloon injury model. Multilevel analyses of gene expression by microarrays, tissues for histology and plasma metabolic measurements provided comprehensive information on the expression of genes of interest.
3.3.3 Mouse carotid ligation model
In Study II, in vivo Pcsk6 -/- mouse model of complete carotid ligation was utilized, which is well-established for investigation of flow-dependent induction of intimal hyperplasia formation, attributable mainly to SMC proliferation392, 393. Briefly, complete ligation of the left common carotid artery close to the bifurcation was performed with prolene non-absorbable suture (Prolene., Ethicon Inc., N.J., USA). This resulted in a turbulent blood flow, defined by low and oscillatory shear stress that initially leads to alterations in the EC alignment and subsequent thrombosis. Typically, sustained obstruction of the flow leads to infiltration of inflammatory cells and SMC activation, which are most prominent up to one week post-injury, followed by the dramatic increase in Ki-67 expression and MMP-9 activation, associated with extensive proliferation and migration, respectively. When thick intima consisting of multiple layers of synthetic SMCs surrounded by ECM components is
Figure 10. Response to vascular injury is characterized by three distinct phases. While early phase signifying acute injury is associated predominantly with cell death, blood coagulation and inflammation, intermediate phase with pronounced, tissue remodeling is characterized mainly by ECM reorganization, SMC activation and phenotypic modulation with increased migration and proliferation. Late stage, corresponding to the re-establishing vessel wall homeostasis,encompasses processes related to cessation of SMC proliferation and reacquisition of contractile features by these cells. (Figure adapted from Röhl et al. JVS Vasc Sci. 2020 Feb 7;1:13-27)
established, rates of both cell division and invasion rapidly decline, which was observed around 2 and 4 weeks timepoints393.
Since this particular model is based predominantly on the SMC response to altered blood flow and, as opposed to rat balloon injury, it does not require endothelial denudation, it is useful for investigating phenotypic modulation of these cells in vascular wall healing from another angle. In addition, with the relatively low cost of both genetic manipulation and housing, mouse models are perfectly suited for the studies of the role of various SMC-specific genes394.