**6. The role of neointimal hyperplasia in graft patency**

graft failure. Injury to the endothelium in addition to surgical manipulation also increases the risk for vasospasm, stenosis, and intimal hyperplasia. Studies have shown that many factors can affect the viability of endothelium; these include temperature, distention, and the compo‐ sition of solution used in vein preparation. Nitric oxide controls vascular tone in addition to causing vasodilatation. Vascular endothelium contains L-arginine which when combined with

guanylate cyclase and subsequently form guanosine 3 prime 5 prime-cyclic monophosphate (cGMP). The cGMP leads to vasodilatation and inhibition of platelet aggregation [19]. Fur‐ thermore, nitric oxide also has been shown to interfere with cell migration, specifically white cells by reducing the adhesion of neutrophils to the endothelial surface. Several cytoprotective properties are conferred through nitric oxide including; scavenging of oxygen free radicals and blocking release of prostaglandin E2 and F2 alpha. These are anti-inflammatory effects, and are quite intricate in detail, but are based on regulation of transcription factors [20], [21]. Nitric oxide also has some cytotoxic effects including decreasing protein synthesis, increasing lipid peroxidation, and decreasing acute phase proteins [22]. Injury to the endothelium directly causes a decrease in nitric oxide release by the endothelial cells and destroys the integrity of the vein. Studies performed by Kown et al. showed that vein grafts treated with L-arginine (nitric oxide is a by-product created when L-arginine is converted to citrulline) can increase

Approximately 12% of patients experience thrombosis of saphenous vein grafts within 30 days of surgery [24]. It has been shown that this acute thrombosis is likely a combination of multiple factors including ischemia and hemostasis during coronary procedures which favors throm‐ bogenesis [25]. The ischemic period in which the vein has been harvested but not yet reimplanted into the myocardium, marks the beginning of the cascade to possible thrombosis. Upon re-establishment of blood flow through the vein it has been shown that neutrophils in the oxygenated blood are attracted to the areas of endothelial injury [26]. This ischemiareperfusion results in a reduction in both basal and stimulated nitric oxide release, yet attenuates the vaso-relaxation responses to the agonist stimulators of endothelial nitric oxide acetylcholine and bradykinin. Together this impairs the release of nitric oxide and down

After the saphenous vein is harvested, the initial injury causes a decrease in nitric oxide due to the traumatic endothelial cell injury from manipulation and distention. Following the ischemic period and after implantation, nitric oxide synthesis will increase due to the reper‐ fusion. Re-implantation causes release of multiple growth factors, and cytokines that cause the migration and proliferation of vascular smooth muscle cells and formation of extracellular matrix into the intimal compartment of the vein graft. Once neutrophils are adherent they initiate further endothelial damage and activation of the coagulation cascade which can lead to thrombosis [1]. The release of nitric oxide at this time can limit neointimal hyperplasia by

. The main target of nitric oxide is to stimulate

nitric oxide synthase forms nitric oxide1

152 Artery Bypass

**5. Reperfusion injury**

levels of nitric oxide and subsequently decrease hyperplasia [23].

regulates nitric oxide production after an ischemic event.

inhibiting this proliferation and promoting apoptosis [27].

Neointimal hyperplasia is the accumulation of smooth muscle cells and extracellular matrix that occurs in the intimal layer of vein. This thickening leads to a narrowing of the lumen and subsequent stenosis of the vein graft. Neointimal hyperplasia is the most widely accepted reason for graft failure at the present time. Many theories exist as to why this occurs but none have been completely proven. Work is currently being performed evaluating the up regulation of genes or proteins that may cause the phenomenon of intimal hyperplasia [15]. Nearly all vein grafts placed into an arterial system develop some areas of hyperplasia within the first four weeks. This acute hyperplasia can narrow the lumen of the vein conduit by as much as 25%.

Many studies have related extensive endothelial injury to neointimal hyperplasia develop‐ ment. Injury can be in the form of extreme venous distention, denudation of the endothelium itself, and degree of vasospasm overcome during harvest [28]. Intimal growth is stimulated by several factors including platelet derived growth factor, transforming growth factor beta, and epidermal growth factor which cause proliferation and subsequent invasion of the smooth muscle cells into the intimal layer [1]. When veins are injured, basic fibroblast growth factor is released from the endothelial cells and smooth muscle cells. This is a very potent mitogen that causes the increased production of multiple regulatory proteins, kinases, and genes that participate in DNA synthesis [29]. The sequential activation and inactivation of the cyclin dependent regulatory kinases (Cdk) leads the smooth muscle cells through the cell cycle [30]. Each cyclin exhibits a cell cycle phase specific pattern of expression with several cell cycle checkpoints at the G1/S station. At these points the kinases interact with a cyclin, specifically D and E interacting with Cdk 4/6, and 2. To progress the cell into the M phase cyclin B is activated. These Cdk proteins are inhibited by activating Cdk 1. The G1 Cdk is part of the retinoblastoma pocket proteins that when phosphorylated can sequester cell cycle regulatory transcription factors. This phosphorylation by retinoblastoma proteins as well as specific cylcin dependent kinases during late G1 leads to activation and release of genes that participate in DNA synthesis. It is this complex cascade of cellular activities that leads to proliferation of smooth muscle cells causing neointimal hyperplasia1, [30]. Further research has shown that other theories also exist as to the mechanism of neointimal hyperplasia that includes a role for perivascular fibroblasts and matrix metalloproteinases (MMP's). It is thought that fibroblasts invade through the media of the saphenous vein graft and differentiate into myofibroblasts. MMP's are the mediators of matrix deposition and degradation, which can cause neointimal hyperplasia. Theories exist that a strategy to avoid hyperplasia would be to use MMP inhibi‐ tors. MMPs compose a super family of 66 known zinc peptidases that degrade collagen, gelatin, and elastin31. MMPs are critical for cell growth and proliferation, cell migration, organ development, reproduction, and tissue remodeling. In all of these biological phenomena, matrix degradation is needed to facilitate changes in cell phenotype. For example, liganddependent cell-matrix associations are critical for modulating cell function, and matrix degradation. These interactions can thereby modulate responses of the cell to its microenvir‐ onment within the saphenous vein.

Vascular smooth muscle cells, monocytes/macrophages, and endothelial cells have all been shown to express MMPs. Vein graft stenosis appears to be associated with increased expression of MMP-9 and increased activation of MMP-2 [32]. Pharmacological inhibitor studies demon‐ strate that MMPs are, indeed, involved in the formation of the neointima. Therefore, with this data it appears that MMPs are critical for smooth muscle cell migration and proliferation, which serve as the cellular basis for neointimal proliferation *in vivo*. Tissue inhibitors of metalloprotienases (TIMPs) are four naturally occurring proteins that inactive MMP's by binding to them. Kranzhofer et al showed that three of these TIMPs are found on saphenous vein grafts [33]. Several regulatory mechanisms exist to keep a precise balance between enzymes that degrade matrix and proteins that inhibit their action. Cytokines and growth factors, specifically platelet derived growth factor BB act together through a protein kinase C dependent mechanism to increase the expression of MMP-9, whereas transforming growth factor-beta and platelet derived growth factor BB induce TIMP-3 expression in vascular smooth muscle cells [31]. However, they do not have any influence on TIMP-1, or TIMP-2 expression. Baker et al. transfected grafts with a gene for TIMP-3 and observed an 84% reduction in neointima at 14 days and 58% reduction at 28 days in porcine vein grafts [34]. This shows promise for a potential preventative treatment of neointimal hyperplasia, but problems such as weakening of pre-existing atherosclerotic plaques need to be addressed and the longer-term benefits of this therapy remain unknown.

expression of biomarkers. These biomarkers include; toll like receptors (TLRs), intracellular adhesion molecules (ICAM), vascular cell adhesion molecule-1 (VCAM-1), and platelet endothelial cell adhesion molecule-1 (PECAM-1). An upregulation of ICAM, VCAM-1, and PECAM-1 was seen in veins that had undergone distention when compared with the nondis‐ tended vein [15]. The expression of these cell adhesion molecules is important because an interaction of VCAM-1 and ICAM-1 with monocytes facilitates the monocytes' recruitment to the vein [36]. Additionally, interactions of ICAM-1 and VCAM-1 with PECAM-1 mediate the process of diapedesis of the monocytes into the vessel wall. These initial cell-mediated events facilitate recruitment of more inflammatory cytokines to the area of injury caused by the damage from distention. PECAM-1 is constitutively expressed on all endothelium regardless

Saphenous Vein Conduit in Coronary Artery Bypass Surgery — Patency Rates and Proposed Mechanisms for Failure

http://dx.doi.org/10.5772/55098

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Toll-like receptors play a very important role in the signaling pathway of inflammation. Traditionally, TLR4 costimulates with CD14 in chronic conditions. Interestingly TLR4 has also been shown to bind directly to lipopolysaccharide without CD14 costimulation, leading to subsequent NF-kappa B activation. Studies in TLR4-deficient mice have shown that despite the presence of lipopolysaccharide, these mice do not develop neointima, suggesting that neointimal hyperplasia is a TLR4-dependent process [15], [37]. TLR4 in cooperation with interleukin-1 receptor plays a significant role in the formation of neointima. TLR4 signaling also promotes a proinflammatory phenotype and plays a role in the early response to vascular injury. Therefore, the upregulation of TLR4 may play a role in the development of graft failure in terms of neointimal hyperplasia. TLR2 activation with MYD88 leads to cytokine production through NF-kappa B pathways. Thus, these data suggest that vein graft failure is likely a multifactorial process that includes neointimal hyperplasia and inflammation. Immediate vein graft failure is most probably due to inflammatory cytokines whereas late failure (1 year after CABG) is due to neointimal hyperplasia. However, the common cause of both of these

**8. The future of prevention: from the research bench to the operating room**

Much interest in reducing neointimal hyperplasia by blocking gene expression is arising. The cell cycle of endothelial cells is now better understood and therefore has allowed for genetics to help play a role in preventing stenosis, thrombosis, and ischemia. If the genetic pathways that are associated with the above process can be fully identified this may ultimately influence coronary graft patency. Ex-vivo work has been promising to show that blocking of the cell cycle via gene therapy has slowed down the atherosclerosis that can lead to graft failure [1].

Repeat coronary vascular procedures will continue to be problematic until an understanding of the mechanisms of vein graft have been elucidated. Thus far, extensive research has been done on this topic, but an overall consensus exists that the saphenous vein is a very fragile and easily injured conduit. Great care must be taken while handling the vein during harvest and preparation to avoid damage or stress to either the external or internal surface of the vein. Avoiding supra-physiologic pressure, prolonged distention periods and manipulations which

processes is quite possibly exacerbated by SV pressure distention [15].

of cytokine activation.
