Fig. 3.1
Vascular response to complete carotid artery ligation. (a–b) Representative cross sections of the left common carotid artery at postoperative day 14 following sham ligation (a, control) or carotid artery ligation (b, ligated). (c) Lesion composition at high magnification (lumen, neointima, media, adventitia), with arrows identifying the external elastic lamina (EEL) and the internal elastic lamina (IEL). (d–e) Representative contralateral right common carotid artery following sham ligation (d, control) or left carotid artery ligation (e, contralateral), with evidence of outward remodeling (e). (f–h) Representative cross sections of ligated vessels from a Rosa26 Cre reporter line [8] crossed with a conditional, inducible smooth muscle myosin heavy chain (SM MHC)-CreERT2 line [9]. The reporter was activated prior to injury to constitutively express LacZ in fully differentiated vascular smooth muscle cells expressing the SM MHC marker [7]. After injury, tissues from vehicle-treated mice (f, control) and tamoxifen-induced mice (g–h) were assayed for β-galactosidase activity, yielding strong staining in the media and neointima, showing that the neointima is predominantly derived from differentiated medial vascular smooth muscle cells. Constrictive remodeling (f) also occurs in human vascular disease and is characterized by decreased circumference of the injured artery in response to decreased flow. Tissue collected by perfusion fixation followed by paraffin embedding and Verhoeff’s staining (a–e) or by perfusion fixation followed by staining for β-galactosidase activity, post fixation, and counterstaining with nuclear fast red (f–h). Scale bar indicates 100 μm (a, b, d–g) or 25 μm (c, h)
In this injury model, medial vascular smooth muscle cells dedifferentiate, proliferate, and migrate across the internal elastic lamina to comprise the majority of neointimal cells present in the lesion [7]. We performed lineage tracing studies that follow the fate of fully differentiated medial vascular smooth muscle cells expressing smooth muscle myosin heavy chain (SM MHC) [7] to estimate the proportion of neointimal cells derived from differentiated SM MHC-positive cells. We crossed ROSA26 Cre reporter mice [8] with conditional, inducible SM MHC-CreERT2 mice [9] and performed tamoxifen induction in 10–14-week-old male mice ending 18 days prior to subsequent carotid ligation. The lesions were allowed to progress for 14 days, harvested by perfusion fixation as described below, rinsed with a Nonidet P-40-based detergent rinse, and stained with X-gal solution. We found that the majority of the neointimal and medial vascular smooth muscle cells were positive for beta-galactosidase activity, indicating that the majority of neointimal cells were derived of medial vascular smooth muscle cell origin (Fig. 3.1f–h).
This review covers the materials needed (see Sect. 3.2), the ligation model (see Sect. 3.3), and a discussion of key points related to the model and the factors that contribute to the remodeling response in this model (see Sect. 3.4).
3.2 Materials
All procedures are performed in compliance with institutional and federal regulations and in accordance with The Guide for the Care and Use of Laboratory Animals by the National Academy of Sciences [10]. A veterinarian should be consulted prior to initiating this injury model. This is a survival surgery that is optimally performed under aseptic conditions. For the anesthesia, ketamine (50 mg/ml) and xylazine (20 mg/ml) are mixed with 0.17 ml of xylazine added per 1 ml of ketamine. The addition of atropine is optional. To anesthetize a mouse weighing approximately 25 g, 50–70 μl of this mixture are injected intraperitoneally with an insulin syringe (0.5 ml, Becton Dickinson). To prevent drying of the cornea during anesthesia, an ophthalmic ointment should be applied (e.g., Lacri-Lube®, Allergan). There are many suppliers of surgical instruments that carry the same or similar tools as those described, and specific descriptions are provided here only as a convenience. Materials needed for this surgical model are summarized in Table 3.1.
Table 3.1
Materials needed for the carotid artery ligation model
Materials for the ligation procedure | Materials for the collection procedure | Materials used for both ligation and collection |
|---|---|---|
Surgery platform, small rubber bands, and tape | Catheter (i.v.), 24-gauge or a 10 mL syringe with a 24–26-gauge needle for fixative perfusion | Dissecting microscope with adequate lighting |
Small hair clipper | Fixative (glutaraldehyde, 4 % paraformaldehyde, or other fixative as desired) | Scalpel |
Betadine solution and 70 % ethanol for skin disinfection | An absorbent underpad for the dissecting surface | Gauze (2 × 2 in.) |
Cotton applicators | Rat tooth forceps | Jeweler’s forceps, straight, 4″ |
6-0 silk suture | Phosphate-buffered saline (PBS) for rinsing the carotids | Microforceps |
Bulldog clamp, Johns Hopkins, 1″ straight | Tissue processing/embedding cassettes | Microdissecting forceps with curved tip |
Bulldog clamp, DeBakey, curved 5″ | Lens paper for wrapping and immobilizing carotids arteries during tissue processing | Scissors, McPherson-Vannas, straight |
Michel wound clip applying forceps 5″ | Carboy with liquid nitrogen | Scissors, additional assorted sizes as desired |
Michel wound clips, 7.5 mm | Dry ice for transfer of flash-frozen specimens | Hemostat, 3″, curved |
3.3 Ligation Model of the Mouse Common Carotid Artery
This model is described in the 1997 publication by Kumar and Lindner [6]. Application of this model to a variety of inbred mouse strains has indicated that the remodeling responses vary depending on genetic background [11]. The surgical procedure is easy to perform and can be completed in less than 5 min from start to finish. This makes this model suitable for studies where large numbers of mice need to be examined.
3.3.1 Surgical Procedure
Following the induction of anesthesia, the mouse is placed in dorsal recumbency on the surgery platform, and rubber bands are used to secure the hind legs to the screws mounted on the side of the platform. Another rubber band is used to hold down the head by the upper incisors. A piece of tape is used to keep the front legs clear from the operating field. The skin along the ventral side of the neck is shaved followed by the application of Betadine and ethanol disinfectant. A midline incision is made along the ventral side of the neck (1.5 cm in length). We routinely perform procedures on the left carotid artery because it has no side branches. The left salivary gland is moved laterally by blunt dissection and held away from the carotid bifurcation by the DeBakey clamp. The pretracheal strap muscles are pushed medially, while blunt dissection of the distal common carotid artery is performed with the microdissecting forceps with curved tips. These forceps are ideally suited to separate the connective tissue from the vessel and place the ligature. The common carotid artery is tied off by placing the ligature just proximal of the carotid bifurcation, with care taken not to disrupt or ligate the vagus nerve. For sham ligation, which may be performed as a surgical control, the common carotid artery is isolated but not ligated. Next, the skin edges are approximated and everted, and the incision is closed with wound clips. The recovery of the mouse on a heating pad is monitored, and postsurgical analgesics and care are administered in accordance with regulations and institutional operating procedures.
3.3.1.1 Surgical Complications
This procedure is generally well tolerated due to redundant blood supply to the brain via the circle of Willis. However, occasionally mice may undergo stroke and/or death following this procedure, especially in cases where there is altered cerebrovascular anatomy with impaired ability to compensate for local flow cessation [12]. Postoperative care should be provided in accordance with institutional and federal guidelines, and appropriate postoperative care may include euthanasia in the case of stroke or impairment. Evidence of nerve damage or stroke may include circling behavior, balance impairment, motor deficits, or spontaneous death. In the absence of other underlying factors, the complications of stroke or death are uncommon. Infection is rare in the carotid artery ligation model although precautions should still be taken to perform the surgery with aseptic technique.
The primary complication associated with this surgery is clotting of the carotid artery, and this can be managed as described in the quantification section.
3.3.2 Perfusion Fixation
For morphometric analyses, perfusion fixation should ideally be performed under physiological pressure with a glutaraldehyde-based fixative, because this abolishes any elastic recoil when the vessel is no longer under pressure. Perfusion fixation via a catheter (24 gauge) placed into the left ventricle is a preferred approach, although a syringe and needle can also be used. If desired, a very small incision can be cut into the left common carotid artery in situ just proximal of the ligature for optimal perfusion prior to fixation. As an alternative to glutaraldehyde, perfusion fixation with 4 % paraformaldehyde can be performed. This is frequently utilized if required for subsequent tissue analysis such as immunostaining.
3.3.3 Tissue Collection and Processing
Careful attention to setup prior to carotid artery harvesting yields the best results for perfusion fixation and collection of carotid arteries for flash freezing.
Procedure for Carotid Artery Collection
Anesthetize the mouse, and confirm anesthesia by loss of toe-pinch withdrawal. Lay the mouse in dorsal recumbency, and spray the neck and chest with 70 % ethanol, which mats the fur and minimizes release of loose fur during subsequent steps. Pinch the skin upward with rat tooth forceps, and cut to remove the skin over the thorax and upper abdomen. Locate the xiphoid process and make lateral cuts from the xiphoid process down the sides of the abdominal cavity. Grasping the xiphoid process upward, carefully poke the scissors into the thorax just lateral to the sternum and cut out the body of the sternum leaving the manubrium intact. Next, cut out the ventral portion of the left ribs to expose the heart. Perform the perfusion fixation step as desired (see Sect. 3.3.2). The whiskers, extremities, and tail will begin to twitch during the perfusion fixation, and the liver will turn from dark red to mottled and light brown. The use of an absorbent underpad over the dissecting surface aids in quick cleanup from this portion of the procedure. After perfusion fixation, the mouse is moved to the dissecting scope platform, in dorsal recumbency with the head proximal to the collector, and the remainder of the skin over the neck is removed. The salivary glands are then pinched upward and cut out, keeping the scissor tips up and dabbing the pooling blood with gauze. Lifting each forepaw in turn allows the clavicle and pectoralis muscles to be cut away from the sternal manubrium in a single cut. Next the sternocleidomastoid muscles are cut away. Then the scissor tips are inserted just below the suprasternal notch, and the manubrium is bisected and then removed by cutting away a portion of the upper ribs on each side to expose the heart. The pretracheal strap muscles are then retracted superiorly, exposing the trachea, followed by sequential removal of the trachea and the underlying esophagus. The carotids are then exposed and cleared. The pericardial fat overlying the base of the heart is pulled upward and snipped, keeping the scissor tips upward to avoid damage to the aortic arch. Using straight forceps, the heart is then flipped and retracted superiorly, allowing a single snip to cut across the ascending aortic arch at the base of the heart. The ascending aortic arch is then grasped with the forceps, and the aortic arch and carotids are removed en bloc, snipping away excess tissue and blotting released blood with gauze. The specimen is freed by snipping at the level of the carotid bifurcation, which will include the suture on the ligated side. The carotids are then rinsed in the PBS and placed in 4 % paraformaldehyde overnight. The following day, the specimens are rinsed in PBS, laid out and wrapped in a single layer of lens paper moistened with 70 % ethanol, and placed in a tissue processing cassette in 70 % ethanol in preparation for tissue processing. The lens paper wrapping allows the carotids and aorta to be placed in anatomical orientation and immobilized during tissue processing. The specimen can then be paraffin embedded on end, with the ligated side on the left, and cross-sectioned at 5 μm, with 5 sections per slide across the 2 mm proximal to the ligature.
Flash-Freezing Protocol
Alternatively, carotids can be harvested and flash frozen for downstream applications of DNA, RNA, or protein analysis. For flash freezing, the perfusion step is performed with PBS rather than fixative, and an effort is made to harvest the carotids as quickly as possible. In the interest of time, the carotids can be separately cut out directly from the aortic arch (left carotid) or from the subclavian artery branch point (right carotid) in situ without first removing the heart. The carotids are then wrapped in small foil packets and dropped into liquid nitrogen, followed by transfer to dry ice for transport to storage at −80 °C.
3.3.4 Morphometric Analysis of Arteries
3.3.4.1 Location
The importance of location in morphometric analysis of vascular lesion formation is well established [6, 13, 14]. Strain differences in neointimal lesion formation should also be considered [11]. In general, the severity of the lesion tends to decrease with distance from the ligature, with the most severe lesion occurring within the first 500 μm of the ligature in wild-type mice [14, 15]. Multiple valid models of quantification have been used, from intermittent sections along the length of the injured artery [13], to sections across a 500 μm–3 mm segment proximal to the ligation [16], to sections at the set distances 2.5, 4.5, and 6.5 mm from the ligation [17], to sections from a single point 3 mm from the ligation [18]. Alternatively, some groups have chosen to obtain measurements from the apex of the lesion as determined by serial sections at 150 μm intervals across the entire length of the artery [19]. Considerations in choosing an appropriate quantification scheme may include whether there is an expected increase or decrease in lesion size in comparison to control, overall cost, and desired number of slides generated and whether the intervening sections are retained for additional analysis.
A quantification scheme that we typically use for lesion analysis includes measurements at the six set distances of 200 μm, 350 μm, 500 μm, 1 mm, 1.5 mm, and 2 mm from the ligature. The rationale is that three measurements are taken 150 μm apart in the proximal 500 μm where the lesion is largest and three measurements are taken at 500 μm apart over the adjacent segment where the lesion tends to start tapering off [14]. The 200, 350, and 500 μm approaches have been previously described in the literature [20], and the additional 1, 1.5, and 2 mm sections ensure capture across a broader section of the lesion. The six measurements can then be used to generate an average lesion size per mouse. The averages can then be compared by student’s t-test, assuming normal distribution of the data, or by analysis of variance (ANOVA), in the case where study design involves comparison of more than two groups. While we have investigated the use of more sophisticated statistical modeling [14] (and Liaw Lab, unpublished data), we find that averaging data for each metric over the six distances is a straightforward and user-friendly method of quantification. Alternatively, depending on the study design, morphometric analysis can be performed at a set distance of 1.5 or 2 mm from the ligature. The advantage of this approach is that it results in less missing data than with models that include the 1 mm proximal to the ligature, the region most prone to clot.
3.3.4.2 Morphometry
The method of fixation and the type of fixative can have a major impact on the morphometric analysis of sectioned vessels. It is important that the vessels be perfusion fixed at physiological pressure. However, intravascular pressure at the time of perfusion is difficult to assess, and with the use of relatively small caliber perfusion catheters, a large drop in pressure is likely to occur along the length of the catheter itself. In addition, if fixatives are used that do not contain glutaraldehyde, the vessel will retain some of its elastic properties which will cause the vessel to contract or shrink upon excision. As a consequence, elastic lamellae will appear wrinkled and wavy, and this can be particularly evident in normal vessels that lack a neointima. The morphometric analysis of sections is facilitated if a histological stain is chosen that highlights the elastic lamellae such as Verhoeff’s stain. This stain makes it particularly easy to trace the internal and external elastic laminae (IEL and EEL) and thus adds to increased accuracy of the measurements. An artery that has undergone constrictive arterial remodeling will display wavy elastic lamellae even if the vessels were harvested following optimal perfusion fixation. With the in situ morphology preserved, glutaraldehyde-based fixatives are ideal when morphometric analyses have a high priority. However, the need to perform immunohistochemistry on vessel sections may require formalin fixation or fresh-frozen tissue.
We typically perform the morphometric analysis by tracing out the lumen circumference, the IEL circumference, and the EEL circumference using a graphics pad, such as the Intuos 4® professional pen tablet by Wacom, in National Institutes of Health ImageJ software [21]. A photo of a stage micrometer at the desired magnification can be used to set the linear conversion rate of number of pixels per mm. This allows determination of the areas within the luminal and elastic laminae perimeter tracings. Tracing of the IEL and EEL should include bisection of the sinusoidal pattern in the case of a wavy line to most accurately capture the circumference and area measurements. The luminal area can be measured directly. The area of the neointima can be determined by subtracting the luminal area from the area bound by the IEL. The medial area is determined by subtracting the area bound by the IEL from the area bound by the EEL. If desired, the tracings can be done by a trained blinded independent observer. Inter-rater reliability tends to be high as long as there are clear guidelines, such as excluding sections where clot is present as the presence of clot can interfere with the determination of the extent of the neointima present (Liaw lab, unpublished data). In this model, the most common outcome measures presented are lumen area, neointimal area, and neointimal/medial (N/M or intimal/medial) ratio, which is determined by dividing the neointimal area by the medial area. N/M ratio is a companion measure of neointimal lesion formation that takes into account changes that may be occurring in the media. Alternatively, some groups prefer to report neointimal lesion formation by measures of intimal thickness and medial thickness [22]. In this scenario, intimal thickness may be measured at the widest point. As neointimal lesion formation is not always concentric or symmetric, we prefer area measurements to thickness measurements. As an additional alternative, numbers of cells in the neointimal and medial layers can be counted and reported. If desired, some groups also present quantification measures for the adventitia [23]. In this case, care should be taken during collection to keep the adventitial layer completely intact.
Morphometric measurements are performed on the ligated side as well as on the unligated side, as the increase in blood flow experienced on the contralateral side can result in outward remodeling of the vessel. For this reason, a sham ligated artery, where the left common carotid artery is isolated but not ligated, is a preferable control vessel for comparison than the contralateral vessel from a ligated animal.
3.3.4.3 Exclusions from Morphometric Analysis
In most cases, the ligated vessels do not show signs of complete thrombotic occlusion with the exception of the 1 mm segment proximal to the ligature. Sections where clot is present are noted but excluded from the morphometric analysis. The percentage of excluded sections can be included in the statistical analysis to rule out statistically different percentages of excluded sections between groups. In rare instances where clotting occurs along a longer segment of the vessel (>2 mm proximal to the ligature), it is usually quite obvious at the time of vessel harvest, and these vessels are excluded from the analysis. Again, these vessels are noted but in our experience do not tend to correlate preferentially with control or experimental groups (Liaw Lab, unpublished data) or with a more or less experienced surgeon.
3.4 Discussion
Features of the vascular response to carotid artery ligation may include a combination of lumen narrowing by neointimal lesion formation and by constrictive remodeling, with relative contributions dependent on factors such as the experimental model used and the genetic background of the mice. Strain-dependent data are available for assistance in choosing an appropriate mouse model for the desired experiment [11]. One major advantage of the ligation model over other models is that it is less likely to be influenced by variables related to surgical technique because it is easy to perform. This fact has made it a very popular model for studying vascular smooth muscle cell dedifferentiation, proliferation, migration, and redifferentiation in vivo [13, 24–38]. In addition, the quick duration of the ligation procedure makes it one of the most suitable models for high-throughput screening.
The type of question to be answered by the experiment will determine when the arteries will be harvested for analysis. In the ligation model, an extensive neointimal lesion will form within 2 weeks. The composition of the neointima, however, will continue to change over the course of several more weeks usually with an increase in extracellular matrix and a decrease in cellularity. Vascular smooth muscle cell proliferation is therefore preferably examined at earlier time points (less than 2 weeks after ligation) and neointimal lesion size preferably at later times (at or after 2 weeks). Typical collection days chosen for this model include an early collection time point (3–7 days), 14 days, and 28 days, although other time points may be used as desired.
In contrast with other denuding arterial injury models, a major inflammatory response is not typical of this model as there is preservation of the endothelium and generally low levels of mechanical injury. Carotid artery ligation would not therefore be an appropriate model for study of the inflammatory response associated with intimal lesion formation unless combined with other atherosclerosis mouse models that might be expected to contribute to the inflammatory response.
Review of the carotid artery ligation literature includes many mediators of the neointimal response, and these have been summarized in Table 3.2. These mediators include the broad categories of adhesion molecules; growth factors, cytokine, and hormone signaling; cytoskeletal; blood pathways/enzymes/blood pressure; reactive oxygen/oxidative stress; secreted proteins/enzymes; cell cycle related; transcriptional regulators; extracellular matrix related/proteases; transmembrane or membrane-anchored signaling molecules; intracellular enzymes; and microRNAs (miRNA). Of particular interest, the contribution of miRNA in neointima formation is a highly dynamic and emerging field. miRNA are small ~18–22 nucleotide noncoding RNA sequences that regulate gene expression. Carotid artery ligation studies focusing on miRNA regulation of intimal hyperplasia are expanding in number, and there is abundant in vitro and in vivo evidence for the role of miRNA in neointimal lesion formation. While the role of miRNA in other models of neointimal lesion formation is outside the scope of this chapter, there are several excellent reviews available on this topic [39–46]. Of note, the use of the carotid artery ligation model in combination with other atherosclerosis mouse models, such as the ApoE null with high-fat feeding or LDL null models, is also beyond the scope of this focused review and thus is not included in Table 3.2.
Table 3.2
Genes involved in the remodeling response after complete ligation of the mouse common carotid artery
Gene target | Description | Phenotype |
|---|---|---|
Adhesion molecules | ||
P-selectin | Null mouse model | Decreased intimal/medial area ratio at 4 weeks, no inflammatory infiltration [13] |
Growth factors, cytokine, and hormone signaling | ||
FGF-2 | Blocking monoclonal antibody | Increased lumen size and vessel diameter at 4 weeks, outward remodeling of contralateral artery [47] |
TNF-alpha | Null mouse model | Decreased intimal lesion at 4 weeks [48] |
p75(NTR) | Null mouse model | Increased intimal lesion and decreased apoptosis at 2 and 4 weeks [34] |
Endothelin (B) receptor | Null mouse model | Increased intimal area, intimal/medial area ratio, and stenosis at 2 weeks [29] |
Fas ligand | FasL-gld mice; point mutation | Increased intimal lesion formation at 4 weeks [30] |
PDGFRbeta | Chimeric mice with PDGFRbeta null cells | At 4 weeks after injury, increased proportion of PDGFRbeta null cells in media and decreased proportion in intima, showing deficiency in migration into intima [49] |
MEKK1 | Null mouse model | Decreased intimal area at 4 weeks [50] |
Akt | Transgenic active Akt1 driven by a VE-cadherin promoter or a minimal SM22α promoter lacking the G/C-rich repressor region | |
IL1beta, IL1R1 | Null mouse models | Loss of either led to reduced intimal/medial area ratios [53] |
Cthrc1 | Transgenic mice with constitutive Cthrc1 expression | Decreased intimal area and intimal smooth muscle cell proliferation at 2 weeks [54] |
S1P receptor2 | Null mouse model | Increased smooth muscle cell replication, yielding large intimal lesions at 4 weeks [55] |
Lysophosphatidic acid receptor | LPA1-null mouse model; LPA1/LPA2 double-null mouse model | LPA1-null mouse had increased intimal area, intimal/medial area ratio, and increased vessel area; LPA1/LPA2 double-null mice had decreased intimal area and intimal/medial area ratio [56] |
Soluble guanylate cyclase | Null mouse model | Selectively in males, sGCα1 nulls have decreased intimal medial area ratio and decreased stenosis [57] |
Protein kinase Cδ | Null mouse model | Loss of PKCδ led to increased intimal/medial area ratio and small luminal area at 4 weeks, associated with decreased apoptosis [58] |
Erythropoietin | i.p. protein injections 3x/week | Epo treatment increased intimal area and intimal/medial area ratio at 4 weeks [59] |
Apelin | Null mouse model | Reduced intimal area and intimal/medial area ratio at 4 weeks [60] |
Wnt4 | Null mouse model | Decreased lesion area and proliferation at 3 weeks [61] |
uPAR | Null mouse model | Decreased medial thickness and intimal/medial area ratio at 4 weeks [62] |
ASK1 | Null mouse model | Decreased intimal area and intimal/medial area ratio at 3 weeks [63] |
Platelet factor 4 | Null mouse model | Decreased total vessel area at 3 weeks [64] |
Cortistatin | i.p. injections of protein every 2 days after ligation; null mouse model | Decreased intimal area with decreased cell proliferation at 4 weeks; null mouse had increased intimal/medial area ratio at 3 weeks [65] |
Histidine carboxylase, histidine receptor | HDC-null mouse; H1R-null mouse | |
IL-19 | Null mouse model | Increased intimal lesion formation at 4 weeks [68] |
Cytoskeletal | ||
Vimentin | Null mouse model | Decreased vessel diameter and medial area at 4 weeks [37] |
ROCK1 | ROCK1+/− mice | Decreased intimal area and intimal/medial area ratio at 4 weeks [69] |
Blood pathways/enzymes/blood pressure | ||
Kallikrein | Adenoviral transduction of human form i.v. | Decreased neointimal lesion formation at 2 weeks, dependent on kinin B(2) receptor [70] |
uPA, α2AP, factor VIII | Null mouse models | |
Tissue factor pathway inhibitor | Intravascular adenoviral delivery of TFPI; TFPI heterozygous mouse | |
Haptoglobin | Null mouse model | Null females had increased intimal hyperplasia only at 8 days, no differences at 20 days [73] |
Vitronectin, PAI-1 | Null mouse models | |
Tissue ACE | Null mouse model | Suppressed medial thickening and less outward remodeling [75] |
C-reactive protein | Human CRP transgenic mouse model | Increased intimal area at 4 weeks [76] |
Thrombomodulin | Recombinant protein i.v. | Decreased intimal lesion and total vessel area in mice receiving thrombomodulin at 4 weeks [77] |
Endothelin1 | Conditional null driven by Tie2-Cre | Decreased intimal area and intimal/medial area ratio at 4 weeks with decreased cell proliferation [78] |
Plasminogen/angiostatin | Plasminogen kringle 1–5 for 4 weeks after injury; angiostatin | Both angiostatin and kringle 1–5 suppressed intimal area and intimal/medial area ratio at 4 weeks [79] |
Reactive oxygen/oxidative stress | ||
Nitric oxide synthase | eNOS or iNOS null mice; eNOS transgenic mice (preproendothelin-1 promoter); NOS2 (iNOS) null mice | Increased intima at 4 weeks in eNOS null; more constrictive remodeling in iNOS null [17, 27]; iNOS null males had decreased intimal area at 4 weeks; females only showed this effect if ovariectomized [80]; loss of NOS2 (iNOS) increased intimal lesion area and intimal/medial area ratio at 4 weeks [81]; decreased intimal and medical areas at 4 weeks in eNOS transgenics [16]; null nNOS or eNOS increased intimal/medial area ratio, while null iNOS did not, and the triple null mice had further increased intimal/medial area ratio [82] |
Cyclophilin A | Null mouse model and conditional transgenic mouse model activated by SM22α-Cre | On null background, decreased intimal and medial areas, and decreased intimal/medial area ratio; transgenic overexpression led to increased intimal and medial areas, and increased intimal/medial area ratio at 2 weeks [83] |
Sequestosome1 | Null mouse model | Increased intimal area and stenotic ratio (percent intima within the total IEL area) [84] |
Secreted proteins/enzymes | ||
Uteroglobin | Adventitial adenoviral delivery of uteroglobin | Uteroglobin decreased intima/media area ratio at 30 days [85] |
Endothelial lipase | Null and transgenic overexpressing mouse models | Null had decreased and transgenic had increased intimal area and intimal/medial area ratio at 2 and 4 weeks [86] |
Cell cycle related | ||
p130 | Null mouse model | Increased intimal lesion and overall vessel wall area [33] |
Skp2 | Null mouse model | Reduced intimal area at 4 weeks [87] |
BubR1 | Hypomorph mouse (20 % of normal) | Decreased intimal area and intimal/medial area ratio at 4 weeks [88] |
Transcriptional regulators | ||
Steroid receptor coactivator-3 | Null mouse model | Increased intimal area and intimal/medial area ratio at 4 weeks [89] |
TR3 | Transgenic expressing dominant-negative TR3 or full-length TR3 (SM22α promoter) | Dominant-negative TR3 led to increased intimal lesion formation; transgenic expression of full-length TR3 inhibited intimal lesion formation [90] |
NF-κB | p105 null mouse model or adeno-associated viral transduction of IκBα into tunica media 2 weeks prior to injury; p50 null mouse model | |
Klf4 | Klf4loxP allele activated by ERT-CRE or Tie2-Cre | |
YAP | Conditional heterozygous mouse model driven by SM22α-Cre | Reduction in intimal area and intimal/medial area ratio at 3 weeks [96]
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