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Physiol. Genomics 25: 286-293, 2006. First published January 24, 2006; doi:10.1152/physiolgenomics.00135.2005
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Received 10 June 2005; accepted in final form 17 January 2006.
Physiological Genomics 25:286-293 (2006)
1094-8341/06 $8.00 © 2006 American Physiological Society

Linkage analysis of neointimal hyperplasia and vascular wall transformation after balloon angioplasty

Andrea L. Nestor1,3, George T. Cicila2, Seth E. Karol1,3, Kay M. Langenderfer1,3, Stacy L. Hollopeter1,3 and David C. Allison1,2,3

1 Departments of Surgery, Pharmacology, Metabolism, and Cardiovascular Science, Medical University of Ohio, Toledo, Ohio
2 Physiology, Pharmacology, Metabolism, and Cardiovascular Science, Medical University of Ohio, Toledo, Ohio
3 Medical University of Ohio Microscopy Imaging Center, Medical University of Ohio, Toledo, Ohio


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Neointimal hyperplasia (NIH), a result of vascular injury, is due to the migration and proliferation of smooth muscle cells through the media and internal elastic lamina leading to vascular occlusion. We used a rat model to find the genetic regions controlling NIH after endothelial denudation in two divergent inbred strains of rats. The Brown Norway (BN) and spontaneously hypertensive rat (SHR) strains have a 2.5-fold difference in injury-induced NIH. A population of 301 F2 (SHR x BN) rats underwent a standard vascular injury followed by phenotyping 8 wk after injury to identify quantitative trait loci (QTL) responsible for this strain difference. Interval mapping identified two %NIH QTL on rat chromosomes 3 and 6 [logarithm of odds (LOD) scores 2.5, 2.2] and QTL for other injured vascular wall changes on rat chromosomes 3, 4, and 15 (LOD scores 2.0–4.6). Also, QTL for control vessel media width (MW) and media area (MA) were found on chromosome 6 with LOD scores of 2.3 and 2.5, suggesting that linkage exists between these control vessel parameters and NIH production. These results represent the first genetic analysis for the identification of NIH QTL and QTL associated with the vascular injury response.

quantitative trait loci; restenosis; vascular injury; inbred strains; Brown Norway rat; spontaneously hypertensive rat


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
INTERVENTIONS IN THE CARDIOVASCULAR system such as arterial grafts, stents, and balloon angioplasties often fail because of the development of neointimal hyperplasia (NIH). There are no effective therapies to prevent or treat this complication. For example, restenosis secondary to NIH occurs in 30–40% of patients after balloon angioplasty for coronary artery disease (27). Drug-eluting stents reduce the occurrence of short-term NIH to 20–30%, but the long-term efficacy of these devices is not known (27). NIH is also associated with the development of significant restenosis in 20% of patients undergoing carotid endarterectomy (1) and is a major contributor to graft failure in coronary and peripheral arteries (24).

NIH is triggered by vascular endothelial cell (VEC) disruption and injury that leads to smooth muscle cell (SMC) migration to subendothelial injury sites, followed by SMC proliferation and apoptosis. Significant neointimal thickening and decreased lumen areas are seen within 2 wk after vascular injury (16, 32). However, vascular occlusion can continue after 2 wk as SMC size increases (16, 32) and proteoglycan matrix deposits continue to thicken the neointima (8, 28, 36, 37, 45). Many factors are involved in, and possibly responsible for, genetically determined variations in the NIH injury response between different strains of animals and among individual patients (1). We postulate that heritable variations in the genes for these vascular injury response elements are possible candidates for the quantitative trait loci (QTL) governing the differences in postinjury NIH among different individuals and animal strains.

We utilized a rat model for NIH formation after vascular injury to genetically dissect this complex biological process (1). In this model, we create a vascular injury by denuding the VECs of the vessel wall, similar to injury occurring in human vessels after cardiovascular interventions. The objective of the present work was to identify chromosomal regions containing the gene(s) responsible for the differences in NIH formation after vascular injury, previously observed between inbred spontaneously hypertensive rat (SHR) and Brown Norway (BN) rat strains (1). We performed a genome scan on a segregating population of 301 F2 (SHR x BN) rats that identified multiple QTL for postinjury NIH and other vascular wall phenotypes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals.
Inbred SHR and BN rats were obtained from Harlan Sprague-Dawley (Indianapolis, IN) and were maintained in the Division of Laboratory Animal Medicine at the Medical University of Ohio, where the genetic crosses were bred. Our previous experiments suggested that the alleles responsible for NIH growth were inherited in a codominant manner (1), and thus we selected an F2 (SHR x BN) intercross population for the genome scan. Female SHR and male BN rats were intercrossed to produce F1 (SHR x BN) progeny that were then used to breed an F2 (SHR x BN) population of 301 males. Reciprocal crosses were not used to breed the initial F1 population because of difficulties in BN rat husbandry. Therefore, the Y chromosome is inherited from the BN strain whereas the X chromosome is equally inherited from both strains. Male SHR and BN rats were also concomitantly raised for comparison. Rats were weaned at 28 days and fed a standard rat chow (Ralston Purina, Diet 5001) with ad libitum access to water.

Vascular injury.
Surgery was performed on male rats at 11–12 wk of age (250–300 g) as previously described (1). Briefly, rats were anesthetized intraperitoneally with 80 mg/kg ketamine and 12 mg/kg xylazine, and the inner left thigh was shaved and disinfected. The skin over the femoral artery was opened, and the femoral artery was isolated by blunt dissection, dilated with lidocaine, and cannulated with a 2-Fr balloon catheter (1, 4). The 2-Fr balloon catheter was advanced to the distal aorta, inflated with 0.1 ml of normal saline, retracted, and deflated until slight resistance was encountered while being withdrawn through the iliac artery. This process was repeated three times to ensure complete endothelial denudation. Finally, the left femoral artery was ligated, and the tissue layers and skin were approximated with an absorbable suture. This procedure produced the "standard vascular injury." This animal study was approved by the Medical University of Ohio Institutional Animal Care and Use Committee and complied with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Pub. No. 86-23, revised 1985).

Perfusion-fixation and preparation of vessel sections.
Rats were anesthetized as above, and the body weight of each rat was recorded at the appropriate time intervals after surgery. A standard perfusion-fixation technique was performed as previously described (1). Primary agarose embedding was used before tissue processing and paraffin embedding to ensure an exact perpendicular orientation of the fixed vessels to the microtome blade. Each harvested vessel was "pinned out" on a straight axis in small, agarose-filled chambers. Once the agarose set, the embedded vessels were cut at the midpoint of the artery and submitted to standard paraffin processing. The "true" perpendicular axes of the midiliac arteries were then placed in blocks for sectioning. Midiliac artery sections were then stained with Verhoeff-van Gieson stain for elastin.

Vessel measurements.
Image analysis measurements of the media area (MA), neointimal area (NIH area), and internal elastic lamina (IEL) length were made on perpendicular cross sections of control and injured arteries. These parameters were used to calculate the percentage of total arterial wall area due to neointimal growth (designated as %NIH), MA, media width (MW), circular area (CA), and lumen size (LS) of each vessel (1). Body weight-adjusted heart weight (BWadj-HW) was calculated by adjusting heart weight for differences in body weight, using the regression of heart weight on body weight for each animal (14, 21). Because lumens of sectioned vessels were sometimes partially collapsed, the CA was calculated with the length of the IEL directly measured by image analysis. CAs were calculated by the formula CA = (IEL)2/4{pi}. To correct for asymmetric neointimal growth in the injured vessels, injured vessel lumen size (inj-LS) was calculated by subtracting the neointimal area of each vessel from its CA: inj-LS = CA – neointimal area. As control vessels did not have a neointimal layer, their CA and LS were identical. The MA of each vessel was directly measured and, in conjunction with IEL length, allowed calculation of MW as follows:

Formula 1(1)
where r2 is the radius of the entire vessel and r1 is the radius of its CA.

Formula 2(2)
Therefore,

Formula 3(3)
Because r1 = IEL/2{pi},

Formula 4(4)
In addition to BWadj-HW, 10 vascular parameters were measured: parameters related to vascular injury include injured vessel CA, injured vessel MA, injured vessel MW, injured vessel NIH area, injured vessel MA + NIH area, injured vessel LS, and injured vessel %NIH. We also measured parameters in the control vessels that were not related to the vascular injury, including control vessel CA, control vessel MA, and control vessel MW.

Statistical analysis.
Distributions of each phenotype in the F2 (SHR x BN) population were assessed for normality with the SPSS computer program (SPSS, Chicago, IL) by skewness, kurtosis, and the Kolmogorov-Smirnov test. Of the phenotypes measured, two required transformation for normality as follows: %NIH values were transformed by taking the square root and BWadj-HW values were normalized by removing outliers from Box Plot analysis and then taking 1 divided by the value (1/X). Both of the transformed phenotypic values (%NIH and BWadj-HW) were found to have a normal distribution by the Kolmogorov-Smirnov test. Many of the distributions were nearly (98.8%) normal and only required removal of outliers as determined by Box Plot analysis. Furthermore, statistical analyses of differences and/or associations between these parameters measured in the parental and F2 (SHR x BN) rats were also analyzed with one-way ANOVA, {chi}2 and a nonparametric statistical method to test for possible associations between these parameters without assumptions about their underlying distribution.

Genotyping with PCR.
Microsatellite loci were genotyped by PCR amplification. Genomic DNA was prepared from the unfixed livers of BN, SHR, and F2 (SHR x BN) rats isolated with a kit (Dneasy 96-well kit, Qiagen, Valencia, CA). PCR amplification was performed with a PTC-100-96Ag thermocycler (MJ Research, Waltham, MA) as previously described (53). PCR products were electrophoretically size-fractionated on 4% 3:1 (Amresco, Solon, OH) or 4% Metaphor (Cambrex Bio Science, Walkersville, MD) agarose gels and visualized with ethidium bromide staining.

Identification of putative QTL by interval mapping.
Putative QTL were first identified by selective genotyping of a portion of the F2 (SHR x BN) population. Specifically, the highest (n = 45) and lowest (n = 45) 15% of the population with regard to NIH formation were genotyped with polymorphic microsatellite markers, spaced on average 13–25 cM apart (see Supplemental Table S1, which is available at the Physiological Genomics web site).1 The Map Manager QTX computer program (35) was used to order genotypic data into linkage groups, perform interval mapping, and identify putative QTL (29, 44). Potential errors in typing, i.e., loci involved in double-recombination events, were retyped to confirm or correct the results. Threshold values for suggestive linkage of a locus to a QTL were as defined by Lander and coworkers (29, 30), corresponding to the expectation of one false positive per genome scan. Each rat chromosome (RNO) that contained at least one marker suggesting linkage in this %NIH-directed genome scan (RNO2–RNO7, RNO15, and RNO20) was genotyped for the remaining 211 F2 (SHR x BN) rats in the population. Additional microsatellite markers were genotyped in chromosomal regions identified as containing putative QTL in the first phase of the genome scan. Map Manager QTX (35) was used to organize interval maps of the chromosomal regions containing NIH QTL.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Genome scan.
%NIH was measured in the injured vessels of 301 F2 (SHR x BN) rats 8 wk after a standard vascular injury. Initially, 158 markers were used to selectively genotype microsatellite polymorphisms covering rat chromosomes (1–20, X) in the F2 (SHR x BN) population, with the highest (top 15%, n = 45) and lowest (bottom 15%, n = 45) %NIH selected from 301 male F2 rats after the standard vascular injury. Chromosomal regions with logarithm of odds (LOD) scores suggestive of linkage (≥2.0) to the measured phenotype for both control and injured vessel phenotypes, %NIH, MA, NIH area, MA + NIH area, CA, LS, MW, and BWadj-HW, were then selected for genotyping in the remaining 211 rats of the F2 (SHR x BN) population. The average spacing of microsatellite markers was 13.6 cM, with a mean maximum distance of 25.1 cM between markers on a chromosome, resulting in 99.4% of the genome being within 15 cM of a microsatellite marker (see Supplemental Table S1). The resulting map length using the Kosambi correction of all rat chromosomes was 1,780.3 cM, similar to previous estimates (5, 7, 15).

Figure 1 shows a LOD plot of the F2 (SHR x BN) genome scan for the %NIH phenotype based on the top and bottom 15% of postinjury NIH formation (n = 90 rats). This LOD plot for the %NIH genome scan used a free model of inheritance without any assumptions regarding inheritance. Three chromosomal regions showed suggestive linkage to %NIH in this initial screen: 1) RNO2, with a peak LOD score near D2Rat20; 2) RNO3, with a peak LOD score near D3Arb12; and 3) RNO15, with a peak LOD score near D15Rat50 (Fig. 1). QTL for vascular wall measurements of control and injured vessels, as well as for BWadj-HW (RNO2, near marker D2Rat111) were also found in this initial, %NIH-directed genome scan; these include QTL for control vessel measurements unrelated to NIH formation for MA, CA, LS, and MW on RNO2 near marker D2Rat3, MA, CA, and LS on RNO4 near marker D4Rat198, and MA and MW on RNO6 near marker D6Rat44. Furthermore, vascular wall measurements of injured vessel QTL related to NIH growth were found for MA on RNO4 near marker D4Rat198, CA and LS on RNO5 near marker D5Rat20, as well as MA on RNO7 and RNO20 near markers D7Rat20 and D20Rat1, respectively. Complete genotyping of these QTL by chromosome was then performed for the remaining 211 F2 (SHR x BN) rats for RNO2–RNO7, RNO15, and RNO20.


Figure 1
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  		Fig. 1. Logarithm of odds (LOD) plot of the %NIH genome scan quantitative trait loci (QTL) identified with a free mode of inheritance. Results are from the selective portion of the F2 [spontaneously hypertensive (SHR) x Brown Norway (BN)] population (n = 90) of postinjury %NIH analyzed with 158 polymorphic microsatellite markers.

 
Table 1 summarizes the characteristics of the QTL confirmed in the complete F2 (SHR x BN) population of 301 rats, including the locus nearest the LOD plot peak for each phenotype and its mean value for the three genotypes. The mode of inheritance resulting in the highest significant LOD score for each QTL is also given (30). Fourteen QTL were found, of which only ten (injured vessel QTL) were directly related to postinjury NIH production (Table 1). LOD plots for the QTL identified on RNO3, RNO4, RNO6, and RNO15 are shown in Fig. 2, A–D, respectively. Two significant QTL for injured vessel CA (LOD 4.6) and inj-LS (LOD 4.2) were found on RNO3 located near marker D3Arb12 (Fig. 2A). We also found several suggestive QTL related to injured vessel MA, CA, LS, NIH area, and %NIH, primarily residing in the same area of the distal portion of the q arm of RNO3 (Fig. 2A). The RNO3 QTL associated with injured vessel MA (blue) was located near D3Rat227, with a LOD score of 2.0. The RNO3 QTL associated with injured vessel CA (red) were located near D3Rat12 and D3Arb12, with LOD scores of 2.9 and 4.6. Also in the q terminus of RNO3 located near D3Arb12 are QTL for %NIH, injured vessel LS, and NIH area. The %NIH (transformed data, black) and NIH area (nontransformed data, orange) QTL have similar LOD plots peaking near D3Arb12 and LOD scores of 2.5 and 3.1, respectively. Finally, QTL peaks were found near both D3Rat12 (LOD = 2.8) and D3Arb12 (LOD = 4.2) for injured vessel LS (yellow).


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  		Table 1. QTL identified with Map Manager QTX in F2 (SHR x BN) population for %NIH

 

Figure 2
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  		Fig. 2. Individual LOD plots for phenotypes measured in the %NIH-driven genome scan. Linkage for postinjury NIH production and other vessel-wall phenotypes were found on rat chromosomes RNO3 (A), RNO4 (B), RNO6 (C), and RNO15 (D) for the F2 (SHR x BN) population (n = 301). Thresholds differ for the individual chromosomes because of the use of different models of inheritance (30). CA, circular area; LS. lumen size; NIH, neointimal hyperplasia; MA, media area; MW, media width.

 
Suggestive QTL were also identified on RNO4, RNO6, and RNO15 (Fig. 2, B–D, respectively): a QTL for MA + NIH area was found on RNO4, with a peak LOD score of 2.3 near D4Rat79 (Fig. 2B). RNO6 had two suggestive %NIH QTL peaks near D6Rat40 and D6Rat149, respectively, both with LOD scores of 2.2 (Fig. 2C). RNO6 also had QTL for two control vessel measurements, MA and MW, at the distinct D6Rat44 and D6Rat150 loci. The control vessel MA and MW QTL had similar LOD plots with LOD scores of 2.5 and 2.3, respectively (Fig. 2C). An injured vessel MA QTL was found on RNO15 with a LOD score of 2.4 located near D15Rat95 (Fig. 2D).

BWadj-HW and control vessels.
Table 2 shows the mean ± %CV of BWadj-HW and the CA, MA, and MW of BN, SHR, and F2 (SHR x BN) control vessels harvested 8 wk after the vascular injury. The mean BWadj-HW of the hypertensive SHR rats was significantly higher than that of normotensive BN rats (Table 2), with the mean BWadj-HW F2 (SHR x BN) values intermediate to those of the parental strains (P < 0.001, Table 2). Also, SHR control vessels had smaller CA and larger MA and MW than BN control vessels (Table 2). No associations were found between BWadj-HW and control vessel CA of the individual BN and F2 (SHR x BN) rats [P = not significant (NS)]. Furthermore, the F2 (SHR x BN) control vessels had larger CA than SHR control vessels (P < 0.001, Table 2).


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  		Table 2. Vascular parameters measured for control BN, SHR, and F2 (SHR x BN) vessels

 
Injured and control vessels.
In a preliminary time after injury study, BN and SHR vessels were harvested at 0, 2, 4, 7, 18, 23, and 28 days in addition to 8 wk after injury. There was considerable scatter in the measurements of the vessels taken at these earlier time periods. The only significant difference found was a relative increase in the BN vessel NIH area and %NIH at 8 wk after injury (P < 0.05; data not shown). However, injury-induced neointima was observed in both strains only 2 days after the vascular injury, arguing against differences in intimal denudations from the initial injury being responsible for the 8-wk strain differences in neointima production. The IEL in sections of these vessels from the early postinjury time points were also graded in a coded study for the "number of IEL breaks" and "waviness or corrugation" (graded 0–6), a morphology pattern associated with reactive vasospasm (3, 16, 33). Interestingly, the sections of the vessels taken 0–18 days after injury (30 vessels from each strain) revealed that the SHR IEL had more breaks (mean = 14.3, range 2 to >40) than the BN IEL (mean = 8.7, range 0–32, P < 0.025). Surprisingly, the average "wave grade" of the injured BN vessels was 2 (range 0–6), compared with only 0.6 (range 0–3) for the injured SHR vessels (P < 0.001). Together, these results are consistent with the thinner BN vessels (see below, Tables 2 and 3), which are also more fragile than the SHR vessels (11, 23), being more easily distended than the more rigid SHR vessels. Thus the BN vessels had fewer injury-induced IEL breaks. This difference in injury pattern between the two strains, i.e., the numbers of injury-induced IEL breaks, may have led to differences in injury-induced vasospasm (waves) and possibly contributed to the strain variations in neointimal growth.


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  		Table 3. Vascular parameters measured for injured BN, SHR, and F2 (SHR x BN) vessels

 
Injured BN vessels did have higher NIH area and %NIH than injured SHR vessels (Table 3; Ref. 1). In contrast to our earlier results (1), this increased neointima formation was not associated with a smaller LS for the injured BN vessels, which had a 10% increase in CA compared with the noninjured control vessels, compensating for the occlusive effects of the NIH ingrowth (Table 4). However, the NIH ingrowth in the injured F2 (SHR x BN) vessels did lead to a significant decrease in LS compared with control vessels (Table 4).


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  		Table 4. Comparison of parameters measured for control and injured BN, SHR, and F2 (SHR x BN) vessels

 
The F2 (SHR x BN) control vessels were similar to the BN control vessels and showed strong associations between individual vessel CA and MA (P < 0.001) but also resembled the SHR control vessels, with strong associations found between individual MW and MA (P < 0.001). In summary, the control vessels of BN and SHR rats showed strain-specific associations of the CA, MA, and MW, with the control vessels of the F2 (BN x SHR) rats having attributes of both parental strains. Most importantly, for both of the parental BN and SHR rat strains, no associations were found between the individual control vessel CA, MA, MW, and BWadj-HW measurements and any of the parameters measured in the injured vessels of the same rats (P = NS). In contrast, control vessel MW of individual F2 (SHR x BN) rats were significantly related to the injured vessel MA (P < 0.001) and MW (P < 0.001) of the same F2 rats. Furthermore, values of the individual F2 (SHR x BN) injured vessel MA, MW, CA, MA + NIH area, and %NIH were all positively associated (P < 0.001). Finally, the BWadj-HW of individual F2 (SHR x BN) rats also showed a positive association with the injured vessel CA, MA + NIH area, and %NIH (P < 0.001).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Ultimately, we would like to facilitate the discovery of effective therapies to prevent vascular restenosis by taking advantage of the 2.5-fold difference in NIH formation found between the inbred BN and SHR rat strains (1). The genome scan of the F2 (SHR x BN) intercross population identified regions controlling the formation of NIH on RNO3 and RNO6. Surprisingly, the %NIH-directed genome scan led to the discovery of QTL for vascular wall parameters in both control and injured vessels that were not directly related to NIH formation. The injured vessel vasculature changes were associated with QTL for MA, CA, and LS on RNO3 (Fig. 2A), MA + NIH area on RNO4 (Fig. 2B), and MA on RNO15 (Fig. 2D). The control vessel vasculature changes were particularly interesting, with QTL for both MA and MW found in two distinct peaks on RNO6 (Fig. 2C).

Several NIH-related and injured vessel QTL were found on the distal arm of RNO3 (Fig. 2A): a QTL-containing region was identified on RNO3 near marker D3Arb12 for %NIH, NIH area, CA, and LS. In addition, QTL were found on RNO3 for injured vessel MA near marker D3Rat227 as well as LS and CA near marker D3Rat12. We have examined these regions of the rat genome for known genes and are targeting several candidates for further study that may be controlling the NIH injury response.

Initially, vascular injury triggers platelet aggregation and release of PDGF (1, 39), a chemoattractant for SMCs in the media and other inflammatory cells in the circulation and adjacent tissues (39). SMCs migrate through the ECM of the media, pass through the injured IEL, and proliferate and undergo apoptosis at the subendothelial injury site. SMC migration and proliferation are both triggered and modulated by PDGF (32, 48, 54), TGFß (32, 36, 40, 48), endothelial cell-produced heparin (32, 45), estradiol (19), progesterone (31), IGF-I and IGF-II (48), EGF (48), basic fibroblastic growth factor (bFGF) (48), somatomedin C (32), epidermal growth factor (48), IL-1 (48), and enzymes, some possibly related to the renin-angiotensin-aldosterone system (13, 45). ECM macromolecules such as collagen, elastin, proteoglycans, and adhesive glycoproteins are also involved in maintaining vascular wall integrity after injury (17, 28, 36, 37), and signaling cascades induced by changes in these ECM macromolecules can regulate the migration (6, 45, 51), apoptosis (6, 18, 41), proliferation (16, 32, 45), and differentiation (34) of vascular SMCs, VECs, and fibroblasts. Intriguingly, the QTL interval on RNO3 includes matrix metalloproteinase-9 (Mmp9) and cadherin 22 (Cdh22).

The MMP family consists of 17 known enzymes classified as collagenases, gelatinases, stromelysins, and metalloelastases. Mmp9, of the gelatinase subfamily, is located within the NIH QTL region on RNO3 near locus D3Arb12 and is involved in remodeling the vascular ECM (20). MMP enzymatic activation is controlled by inducible nitric oxide synthase (iNOS) signaling. The endothelial nitric oxide synthase (eNOS) isoforms are constitutively active and produce NO in small quantities. eNOS protects the uninjured vasculature by inducing vasorelaxation in response to the shear stress of increased blood pressure (2, 10, 42), inhibits the transvascular migration and proliferation of SMCs and leukocytes (2, 9, 12, 52), blocks the adherence and aggregation of platelets to the vessel walls (2, 9, 12, 38, 52), and promotes endothelial cell growth and proliferation (2). iNOS produces NO in large quantities, is stimulated by vascular damage, and abrogates the vascular protective effects of eNOS-produced NO. NO produced by iNOS thereby promotes injury-induced vasospasm, adherence and aggregation of platelets to the injury site, transvessel migration of SMCs and inflammatory cells (2, 9, 12, 26, 38, 52), and upregulation of MMP expression (26). MMP expression leads to the enzymatic degradation of the ECM and facilitates SMC migration through the media and IEL of the injured vessel.

Another candidate gene in the RNO3 QTL-containing region is a cadherin, Cdh22. Cadherins are adhesion molecules involved in calcium-dependent cell-cell adhesions and may aid the migration of SMCs through the ECM. Cdh22 also participates in a signal transduction pathway regulating expression levels and localization of ß-catenin, which also controls cell proliferation, migration, and apoptosis by the Wnt growth factor pathway (22). We also scanned genes present in the RNO6 QTL-containing region. Although several genes could potentially be involved in the process of NIH formation, none was as obvious a candidate as Mmp9 and Cdh22 for governing the NIH injury response.

We previously found (1) slight decreases in the CA (P = NS) and LS of injured BN vessels compared with noninjured controls. It was possible, however, that this finding was in part due to the noninjured BN vessels being more flexible and difficult to properly orient in the tissue blocks for perpendicular sections than the injured vessels, i.e., the noninjured vessels lacked the rigidity of the injured vessels imparted by the neointima itself and the postinjury inflammatory response. If this was the case, such bias in section perpendicularity would artificially elevate the control vessel CA values. In the present study, we used primary agarose embedding before processing and paraffin embedding to ensure an exact perpendicular orientation of the fixed vessels to the microtome blade (see MATERIALS AND METHODS). A different result from that previously was found for sections prepared in this manner, with a 10% increase of CA of the injured BN vessels occurring, which more than compensated for any occlusive effects of the neointima on the LS of the injured BN vessels (P = NS, Table 1).

BWadj-HW and vessel-wall parameters such as CA, MW, and %NIH are complex phenotypes, presumably controlled by the interplay of multiple genetic and environmental factors. Phenotypes influenced by the same environmental or genetic factors are expected to have expression levels that are positively or negatively linked. Phenotypes without common underlying causal factors would not be expected to show a coordinated pattern of expression. Also, the degree of expression of certain phenotypes before injury might influence the extent, and thereby the subsequent repair, of the standard vascular injury, i.e., the standard vascular injury might produce different degrees of damage to thick- and thin-walled vessels. If this were the case, then the initial MA or MW of a given F2 (SHR x BN) vessel might have a direct bearing on the amount of injury transmitted to that vessel and, in turn, influence its postinjury NIH production. In support of this, the differing numbers of postinjury IEL "breaks" and "waves" between the BN and SHR parental strains also suggest that a genetic difference in vessel fragility and distensibility (11, 23) may directly influence the postinjury responses of the vasculature. We are currently trying to determine whether or not these differences in postinjury vascular changes between the BN and SHR strains can be influenced by endothelial vasodilators.

As the SHR rat is perhaps the best-studied genetic model of hypertension and F2 (SHR x BN) populations are extensively used to identify QTL for blood pressure and related traits (46, 49, 50), elevated blood pressures may have influenced the response to the standard vessel injury in our population of F2 (SHR x BN) rats. Each of the F2 (SHR x BN) rats in our population was phenotyped for BWadj-HW, which shows a strong correlation with blood pressure in rat models (43, 55), including segregating populations of rats (reviewed in Ref. 47). Here we used BWadj-HW as an indirect (and imperfect) measure of the blood pressures observed in the population. As expected, the mean BWadj-HW of F2 (SHR x BN) rats was intermediate to, and significantly different from, the higher values of SHR rats and the lower values of BN rats (Table 2). The lack of association between BWadj-HW and control vessel CA of the individual BN and F2 (SHR x BN) rats and the increased CA observed in F2 (SHR x BN) control vessels, compared with those of SHR control vessels (Table 2), suggest that the mechanism(s) producing small, thick-walled vessels in the SHR rats is not present in the F2 (SHR x BN) rats.

The finding of QTL for non-NIH-related phenotypes, especially those for control vessel MA and MW (Table 1), in the %NIH-driven genome scan supports the possibility of causal connections between certain control vessel parameters and postinjury NIH production. Specifically, the initial genome scan was performed on rats at the extremes of the F2 (SHR x BN) %NIH phenotypic distribution, followed by genotyping of the remaining F2 rats in the chromosome regions where QTL were identified. The complete %NIH-driven genome scan revealed 14 QTL, of which only 10 (injured vessel parameters) were directly related to NIH (Table 1). The non-NIH-related QTL (control vessel parameters) can only be explained by chance or causal connections between postinjury NIH and these phenotypes.

If future studies with congenic strains confirm these associations between the control vessel parameters and postinjury neointima production, these relationships may allow the identification of vessels or individuals with an increased susceptibility to NIH after vascular procedures. Also, development of congenic strains that delimit the chromosomal intervals in which causative gene(s) for %NIH-related QTL reside should facilitate the future identification of the genes responsible for the strain differences in postinjury NIH formation observed between BN and SHR rats. This discovery may, in turn, facilitate the identification of orthologous, NIH-related genes in humans, as disease-specific QTL found in rodent studies have often been shown to be located within orthologous human chromosomal regions containing QTL for the same (or related) phenotypes (25, 56). Ultimately, the detection of NIH QTL may lead to the identification of the gene(s) controlling vascular stenosis and the development of new therapies to prevent this complication.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by American Heart Association Ohio Valley Affiliate Grant 0051562B to D. C. Allison and National Heart, Lung, and Blood Institute Grant HL-68994 to G. T. Cicila, as well as the Cancer Biology Fund of the Medical University of Ohio Foundation.


   ACKNOWLEDGMENTS
 
We thank Dawn Pauli for technical assistance with genotyping and maintenance of the rat colony.


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: A. L. Nestor, Dept. of Surgery, Medical Univ. of Ohio, 3035 Arlington Ave., Toledo, OH 43614-5804 (e-mail: anestor{at}meduohio.edu).

1 The Supplemental Material for this article (Supplemental Table S1) is available online at http://physiolgenomics.physiology.org/cgi/content/full/00135.2005/DC1. Back


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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