Graft Adaptation Research Articles

Control of Blood Vessel Identity: From Embryo to Adult

Arteries and veins have been historically defined by the direction of blood flow and oxygen tension within the vessel, in addition to their functional, hemodynamic, and anatomical differences. It is now known that the molecular identity of these vessels is genetically predetermined, with specific molecular pathways activated during the development of arteries and veins. Eph-B4 is a determinant of venous differentiation and Ephrin-B2 is a determinant of arterial differentiation. Placement of a vein into the higher pressure and flow of the arterial circulation results in adaptation of the vein to the arterial environment. There is selective loss of Eph-B4 expression without induction of Ephrin-B2 expression during vein graft adaptation. These findings suggest that loss of venous identity is the crucial mechanism in vein graft adaptation and that developmentally critical determinants of vessel identity are plastic during adult life.

Venous Identity Is Lost but Arterial Identity Is Not Gained During Vein Graft Adaptation

Ephrin ligands and Eph receptors are signaling molecules that are differentially expressed on arteries and veins during development. We examined whether Eph-B4, a venous marker, and Ephrin-B2, an arterial marker, are regulated during vein graft adaptation in humans and aged rats. Eph-B4 transcripts and immunodetectable protein are downregulated in endothelial and smooth muscle cells of patent vein grafts in both humans and in aged rats, whereas Ephrin-B2 transcripts and protein are not strongly induced. Other markers of arterial identity, including dll4 and notch-4, are also not induced during vein graft adaptation in aged rats. Because VEGF-A is upstream of the Ephrin-Eph pathway, and expression of VEGF-A is induced only at early time points after exposure of the vein to the arterial environment, we inhibited VEGF-A in vein grafts using an siRNA-based approach. Vein grafts treated with siRNA directed against VEGF-A demonstrated a thicker intima-media containing alpha-actin, consistent with arterialization, but did not contain Eph-B4 or Ephrin-B2. Venous identity is preserved in the veins of aged animals, but is lost during adaptation to the arterial circulation; arterial markers are not induced. Markers of vessel identity are plastic in adults and their selective regulation may mediate vein graft adaptation to the arterial environment in aged animals and humans.

Tumor necrosis factor-α and the early vein graft

Tumor necrosis factor-alpha (TNF-alpha) has been implicated in the blood vessel wall response to hemodynamic forces. We hypothesized that TNF-alpha activity drives neointimal hyperplasia (NIH) during vein graft arterialization and that anti-TNF-alpha therapy would inhibit NIH. Rabbits underwent bilateral vein grafting using jugular vein. All distal branches except the occipital artery were unilaterally ligated to create distinct flow environments between the bilateral grafts. Vein grafts were harvested sequentially up to 28 days for TNF-alpha messenger RNA (mRNA) quantitation. In separate experiments, animals received short-term or long-term dosing with pegylated soluble TNF-alpha type I receptor (PEG sTNF-RI) or vehicle. After 14 to 28 days, grafts were analyzed for morphometry, proliferation, apoptosis, and PEG sTNF-RI distribution. Quantitative mRNA assay (TaqMan) revealed shear-dependent (P < .001) and time-dependent (P < .001) TNF-alpha expression. TNF-alpha induction was maximal at day 1 and gradually decreased over time, but was persistently elevated even 4 weeks later (P < .001). Low shear (associated with increased NIH) resulted in significantly higher TNF-alpha mRNA expression (P = .03). PEG sTNF-RI was found in high concentrations in the serum and localized to NIH. The high-flow and low-flow vein grafts from treated animals demonstrated similar volumes of NIH compared with controls. PEG-sTNF-RI had only modest impact on vascular wall cell turnover, as reflected by terminal deoxynucleotide transferase-mediated deoxy uridine triphosphate nick-end labeling (P = .064) and anti-Ki-67 (P = .12) assays. Placement of a vein into the arterial circulation acutely upregulates TNF-alpha; this expression level correlates with the degree of subsequent NIH. Pharmacologic interruption of this signaling pathway has no significant impact on NIH or wall cellular proliferation/apoptosis, suggesting that early vein graft adaptations can proceed via TNF-alpha-independent mechanisms.

Morphological Adaptation of the Bone Graft and Fused Bodies after Non-Instrumented Anterior Interbody Fusion of the Lower Cervical Spine

To assess the remodelling process of the bone graft and fused bodies after non-instrumented anterior interbody fusion with autogenous iliac graft in patients with spondylosis, infections, fractures, or disorders of the cervical spine. 68 patients aged 18 to 58 years who underwent non-plated anterior lower cervical interbody fusion with an iliac graft were retrospectively studied. Diagnoses of the patients were degenerative disc diseases (n=32), disc herniation (n=15), fractures (n=13), and tuberculosis (n=8). The Robinson and Smith technique was used to treat degenerative disc diseases and protruded disc, and the Bailey and Badgley procedure for fractures or tuberculosis of the cervical spine. 34, 25, and 9 patients underwent one-, 2-, and 3-segment fusions, respectively. 18 of the 25 patients underwent two-segment fusion with a single large bone block, and 7 with 2 separate bone blocks for each segment. Four of the 9 patients underwent three-segment fusion with a single large bone block, and 5 used separate grafts for each segment independently. Plain and stress radiography was primarily used to assess the fusion. Computed tomography and magnetic resonance imaging were also used in some patients. Some anterior graft extrusion (amounting to less than 10% of corresponding anteroposterior body width) was used to observe the remodelling during graft-take and thereafter. Postoperative cervical traction for 2 to 4 weeks, then cervical collar immobilisation for 4 to 12 weeks were strictly followed according to the numbers of fused segments. A halo vest was applied in 4 patients with fracture undergoing 3-segment fusion as they could not tolerate the prolonged bed rest or rigid cervical brace. The mean time for the graft to fuse was 8.6 (range, 7-14) weeks in patients who underwent each segment fusion with independent free grafts, and 10 and 14 weeks in those who underwent 2- and 3-segment single large graft fusion, respectively. The final loss of disc height and joint angle were negligible, regardless of the extent of fusion. Bony absorption of the anteriorly protruded part of the graft began at postoperative week 10 (range, 6-28), which coincided with the time of graft-take and initiation of remodelling. The earliest sign of bony absorption of the anteriorly protruded part of the graft indicated the initiation of the graft-take and the graft remodelling. The inwaisting sign of the surgically fused block of vertebral bodies was a morphological adaptation. Despite the altered biomechanics of the spine in the fused area, the inwaisting sign indicated maintenance of normal function at the parafusion motion segments.

Early biomechanical changes in lower extremity vein grafts—distinct temporal phases of remodeling and wall stiffness

The geometric and biomechanical changes that contribute to vein graft remodeling are not well established. We sought to measure patterns of adaptation in lower extremity vein grafts and assess their correlation with clinical outcomes. We conducted a prospective, longitudinal study of patients undergoing infrainguinal reconstruction with autogenous conduit. In addition to standard duplex surveillance, lumen diameter (of a defined index segment of the conduit) and pulse wave velocity (PWV) were assessed by ultrasound imaging at surgery and at 1, 3, and 6 months postoperatively. Graft dimensions and wall stiffness were correlated with clinical outcomes. There were 92 patients and 96 limbs in this study. On average, vein graft lumen diameter increased during the first month of implantation from 0.37 +/- .01 cm to 0.45 +/- 0.02 cm (mean +/- SEM; P = .002), representing a relative change of +21.6% (median +/- 14%; range, -31 to +67%) during this period. Of the entire cohort, 72% of grafts demonstrated appreciable dilation of the index segment during the first month. Index segment lumen diameter did not change appreciably beyond 1 month, with the notable exception of arm vein conduits, which showed continued tendency to dilate. PWV increased during the first 6 months (17.2 +/- 1.2 m/s to 23.2 +/- 2.4 m/s; P = .008), reflecting a nearly 40% increase in conduit stiffness (2.0 +/- .6 Mdynes/cm to 3.3 +/- .8 Mdynes/cm, P = .01). The greatest relative increase (25%) in PWV occurred from months 1 to 3. Loss of primary patency occurred in 24 cases (19 revisions, 5 occlusions), with a mean reintervention time of 7.6 months. Grafts that demonstrated early positive remodeling (lumen dilatation) had a trend of increased primary patency (P = .08, log rank). Among the grafts that failed, a trend was noted toward greater wall stiffness at 1 month, 2.7 vs 1.5 Mdynes (P = .08). Vein graft remodeling appears to involve at least two distinct temporal phases. Outward remodeling of the lumen occurs early, and wall stiffness changes occur in a more delayed fashion. Early outward remodeling may be important for successful vein graft adaptation.

Magnification Endoscopy as a Reliable Tool for the Early Diagnosis of Rejection in Living Related Small Bowel Transplants: A Case Report

The purpose was to determine whether magnification endoscopy (ME) accurately diagnosed rejection in living related small bowel transplants (LRSBTx) during initial morphological adaptation of segmental intestinal grafts. The small bowel recipient was a 44-year-old woman with short gut syndrome following multiple bowel surgeries for familial adenomatous polyposis. ME was enhanced by chromoendoscopy staining. Bowel mucosa was washed with acetic acid and stained with methylene blue for optimal visualization of mucosal villi and to improve the diagnostic yield of biopsies. The recipient underwent surveillance ME with biopsy 16 times through the ileostomy in the first 9 months following transplantation. The recipient developed diarrhea in the postoperative course, which led to the suspicion of rejection. ME findings of patchy villus blunting were consistent with biopsy samples that showed mild acute cellular rejection. Episodes of rejection were treated with high-dose immunosuppressants and steroids. Reversal of rejection was monitored by follow-up ME, which showed increased length of villi and normalization of morphology. Biopsy confirmed these findings. The first endoscopy, at 5 days posttransplant, showed no evidence of intestinal ischemia. LRSBTx involves early morphological adaptation of the recipient small bowel mucosa, characterized by an increased length of villi. ME is a reliable technique to follow adaptation and detect early rejection. The superior imaging of small bowel mucosa created by ME chromoendoscopy enables early diagnosis and delivery of more prompt antirejection therapy to prevent progression of rejection. ME also confirmed that segmental LRSBTx caused minimal ischemic injury to the recipient.

Is the increase in type III collagen of the patellar tendon graft after ligament reconstruction really caused by “ligamentization” of the graft?

To test the hypothesis that extrinsic cells that infiltrate the devitalized patellar tendon (PT) synthesize type III collagen even in the environmental milieu of the native PT, we conducted the present experimental study using the rat in situ frozen-thawed PTs. Tissue culture showed no cell outgrowth from the tendons immediately after the freeze-thaw treatment. Analysis by RT-PCR showed that the expression level of type III procollagen mRNA in the frozen-thawed tendon was significantly higher than that in the sham-operated tendon at 6 and 12 weeks. Immunohistological findings showed positive type III collagen staining around cells that had infiltrated the necrotized tendon at 3, 6, and 12 weeks. In addition, the elastic modulus of the in situ frozen-thawed tendon at 6 weeks was significantly less than that of the sham-operated tendon. The present study indicates that extrinsic cells that had infiltrated the devitalized PT synthesized type III collagen at least for 12 weeks even in the environmental milieu of the native PT. These findings raised the question whether the increase in type III collagen of the PT graft after ACL reconstruction is really caused by "ligamentization," the adaptation of the PT graft to the ACL environment.

Cellular, molecular and immunological mechanisms in the pathophysiology of vein graft intimal hyperplasia

Coronary artery disease, leading to myocardial infarction and ischaemia, affects millions of persons and is one of the leading causes of morbidity and mortality worldwide. Invasive techniques such as coronary artery bypass grafting are used to alleviate the sequelae of arterial occlusion. Unfortunately, restenosis or occlusion of the grafted conduit occurs over a time frame of months to years with a gradual reduction in patency, especially in vein grafts. The events leading to intimal hyperplasia (IH) formation involve numerous cellular and molecular components. Various cellular elements of the vessel wall are involved as are leucocyte-endothelial interactions that trigger the coagulation cascade leading to localized thrombus formation. Subsequent phenotypic modification of the medial smooth muscle cells and their intimal migration is the basis of the lesion formation that is thought to be propagated by an immune-mediated reaction. Despite intense scrutiny, the pathophysiology of IH remains an enigma. Although several growth factors, cytokines and numerous other biomolecules have been implicated and their relationship to prohyperplasia pathways such as the phosphatidyl-inositol 3-kinase (PI3K)-Akt pathway has been established, many pieces of the puzzle are still missing. An in-depth understanding of early vein graft adaptation and progression is necessary to improve the long-term prognosis and develop more effective therapeutic measures. In this review, we have critically evaluated and summarized the literature to elucidate and interlink the numerous established and emerging factors that play a key role in the development of IH leading to vein graft restenosis.

Early Differential MMP-2 and -9 Dynamics During Flow-Induced Arterial and Vein Graft Adaptations

Arteries and vein grafts respond differently to reductions in flow, with arteries demonstrating inward remodeling through only limited structural reorganization of the media and vein grafts developing a thickened intima, with little change in the external diameter. In an effort to mechanistically explore the biology of this contrasting behavior, we hypothesized that this differential response in flow-mediated remodeling is driven by unique temporal expression patterns and functional activities of the matrix metalloproteinase (MMP)-2 and -9, key effectors of blood vessel architecture remodeling. In rabbits, bilateral carotid vein grafting was coupled with unilateral partial distal ligation to create a sevenfold flow differential between sides. Vein grafts and proximal carotid arteries were then harvested for morphological and biochemical studies at time points ranging from 1 to 14 days. Vein grafts demonstrated an early, transient increase in pro-MMP-9 within adherent monocytes and endothelial cells. This was followed by a delayed increase in smooth muscle cell active MMP-2, which was coincident with the onset of intimal thickening at days 7 and 14 and significantly elevated by low flow. In contrast, arteries showed no elevation in pro MMP-9 above baseline, but demonstrated a transient increase in latent MMP-2 preceding the flow-mediated change in vessel diameter. These data provide evidence for unique and independent roles of MMP-2 and -9 in the process of hemodynamically driven vascular remodeling.

Innovative Approaches to Improving Organ Availability for Small Bowel Transplant Candidates

Small bowel transplant candidates have unique features that create special challenges for providing them with organs in a timely manner. More than half of intestinal transplant candidates also need livers. Because there is a greater overall demand for livers than for small bowels, organ allocation in this candidate subset is dictated by the liver allocation policies. However, the extremely high mortality rate associated with patients on the waiting list for combined liver-intestinal transplantation (LIT) attests to the inadequacies of the liver allocation system in recognizing the unique risk factors in patients with intestinal failure who develop end-stage liver disease (ESLD). This report addresses some of the organ allocation issues underlying the discrepancies in waiting list outcomes seen in LIT candidates and discusses possible contributory factors and potential solutions to this problem. The use of living donor and segmental grafts from deceased donors are discussed specifically as potential surgical solutions to the problem, and the potential utility, limitations, and ethical concerns associated with these innovative strategies are discussed.

Further evidence that epidermal growth factor enhances the intestinal adaptation following small bowel transplantation

Recent reports indicate that epidermal growth factor (EGF) plays a crucial role for graft adaptation in rat model of small bowel transplantation (SBT). The administration of EGF enhances intestinal cell proliferating rate and the recovery of mucosal structure. However, the effect of EGF on biological functions including glucose absorption in intestinal graft remains to be elucidated. SBT was performed in the two-step procedure. On the first step, intestinal graft (30-cm jejunum) from Brown Norway rats was exteriorized through abdominal wall as a Thiry-Vella loop in recipient Lewis rats for one week. On the second surgery (POD 7), recipient jejunum was replaced orthotopically by the graft, and transplanted rats were treated intraperitoneally with EGF or its vehicle for 3 days. Analyses of histology and biological functions in the graft were done at POD 14. EGF increased both levels of villus height and crypt depth in the graft of transplanted groups. EGF enhanced the glucose absorption as well as the induction of sodium glucose cotransporter 2- to 3-fold in transplanted groups. Further, EGF stimulated the activities of disaccharidase (maltase and sucrase) and the induction of dipeptide cotransporter. These results demonstrate that EGF enhances the structural and functional adaptation of intestinal grafts after SBT. EGF may be useful therapy for patients following intestinal transplantation.

Poster 9: An alternative method of platelet rich plasma preparation

Statement of the Problem: Platelet-rich plasma (PRP), produced from autologous blood is currently used as an adjunct to bone grafting. Platelets are enriched by centrifugation and clotted with bovine thrombin, which has been associated with the development of antibodies to clotting factors V, XI, and thrombin, resulting in the risk of life-threatening coagulopathies. Thrombin causes significant shrinkage of PRP, raising concern about graft adaptation as well as the potential loss of growth factors from the graft composite. Thrombin signaling of platelets is mediated by a G protein-coupled protease-activated receptor (PAR) which is activated after thrombin binding. The amino terminal end of PAR is cleaved resulting in a new amino terminus which acts as a tethered ligand and binds intramolecularly to the body of the PAR, causing a transmembrane signal. Synthetic peptides such as thrombin receptor activator peptide-6 (TRAP) activate the receptor independent of receptor cleavage. In this report we evaluate the potential of TRAP and TRAP/bone substitutes in minimizing clot retraction and promoting maximum graft adaptation.

Wall shear modulation of cytokines in early vein grafts

Clinical relevance Neointimal hyperplasia continues to limit the durability of vein bypass grafts. Emerging evidence suggests that inflammatory mechanisms drive the neointimal hyperplasic response. This study demonstrates that specific hemodynamic forces (altered wall shear stress) differentially affect early pro-inflammatory interleukin (IL)–1β and delayed anti-inflammatory IL-10 signaling. These distinct temporal windows for IL-1β and IL-10 cytokine expression offer strategies for appropriately timed pro-inflammatory and anti-inflammatory therapies to interrupt pathologic vein graft adaptations.

Early adaptation of human lower extremity vein grafts: wall stiffness changes accompany geometric remodeling

Objective To quantitatively describe the temporal changes in elastic properties and wall dimensions in lower-extremity vein grafts after implantation. Design of study This is a prospective study of patients (N = 38) undergoing lower extremity bypass grafts (N = 41) with autologous veins. Pulse wave velocity (PWV), luminal diameter, and wall thickness measurements were obtained by duplex ultrasound scan intraoperatively and at 1, 3, and 6 months postoperatively for assessment of graft dimensions and wall stiffness. Results Lower extremity vein grafts showed an increase in PWV (from 16 ± 1 to 21 ± 3 cm/s; mean ± SEM; P = .08), reflecting an increase in wall stiffness (from 1.2 ± 0.2 to 2.5 ± 0.7 × 10 6 dynes/cm; P = .02) and wall thickness (from 0.47 ± 0.03 to 0.61 ± 0.004 mm; P = .04) over the first 6 months after implantation. Changes in lumen diameter were positively correlated with changes in external graft diameter ( P < .01) and negatively correlated with initial lumen diameter ( P < .01) but not with changes in the wall thickness. Conclusions These results suggest complex remodeling of vein grafts during the first several months after implantation, with increased wall thickness occurring independent of variable changes in lumen diameter. Simultaneously, a marked increase in wall stiffness over this interval suggests a likely role for collagen deposition.

Recent Progress of Vascular Graft Engineering in Japan

The development of a small-diameter vascular graft has long been awaited. This review covers research activities, achievements and progress on vascular engineering in Japan, which was conducted over the last decade. The article includes recently developed experimental scaffolds, biologically active artificial extracellular matrices (ECMs) or non-fouling synthetic coatings, cell sourcing including the autologous vascular cell type, endothelial progenitor cells and genetically-engineered, temporary endothelial-like cells. The discussions were presented from biomechanical, biomaterial, cellular and tissue aspects. Once the mechano-biological and biologically active extracellular milieus are established in a designed vascular graft, the functional, structural and mechanical tissue morphogenesis and adaptation of implanted vascular grafts may proceed with implantation duration, and the spatio-temporal tissue modulations at cytokine, cellular, ECM levels under physiological stress proceed to regenerate vascular tissue architecture. The ultimate solution to a small-diameter vascular graft should be realized by optimal combinations of these factors.

Mechanisms of graft rejection and adaptation in pediatric liver transplantation

Mechanisms of graft rejection and adaptation in pediatric liver transplantation

Antisense basic fibroblast growth factor alters the time course of mitogen-activated protein kinase in arterialized vein graft remodeling.

Neointimal hyperplasia (NIH) is complete by 3 weeks in rabbit vein grafts implanted into the arterial circulation. Activation of the mitogen-activated protein kinase (MAPK) family of protein kinases is thought to be critical in remodeling events such as cellular proliferation, differentiation, and migration, as found in NIH. We previously demonstrated that antisense basic fibroblast growth factor (ASbFGF) inhibited the synthesis of basic fibroblast growth factor (bFGF) in the balloon injury model of NIH. We examined the effect of ASbFGF on NIH and the time course of MAPK, extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal protein kinase (JNK), and p38 kinase activation in arterialized vein grafts. Carotid interposition of a vein bypass graft was performed in 75 New Zealand White rabbits. Segments of the external jugular vein were transfected with a replication-deficient adenovirus containing the messenger RNA sequence for rat ASbFGF at 1 x 10(10) plaque-forming units per milliliter; control animals were given phosphate-buffered saline solution (PBS) alone. Rabbits were killed at 30 minutes, 4 days, 7 days, and 21 days (n = 8). Four grafts in each group were fixed with formalin and embedded in paraffin, then processed with elastin-collagen and hematoxylin-eosin stains. The other four grafts were individually frozen, and total protein was extracted. Phosphorylation of MAPK, ERK1/2, JNK, and p38, was determined with Western blot analysis and immunohistochemistry. Groups were compared with analysis of variance. The thickness of neointima in the PBS group and the ASbFGF group at 21 days was 60.2 +/- 2.1 and 39.4 +/- 2.1 microm, respectively (P <.01). In both the control and ASbFGF groups, all 3 MAPKs demonstrated activation compared with preimplantation levels. However, when compared with the PBS group the ASbFGF group showed greater than 33% inhibition of all three MAPKs by day 4 and day 7 (P <.05), but no significant difference in any MAPK activation by day 21 (P >.05, all groups). Cells staining positive for activated MAPK were found in the neointima and adventitia of vein grafts in both the PBS and ASbFGF groups. MAPKs are activated during the first week after vein graft implantation. Grafts treated with ASbFGF demonstrated reduced MAPK activation and less neointimal thickening. These results suggest that the process of vein graft adaptation to the arterial circulation, and subsequent NIH, may depend on basic fibroblast growth factor activity, which is mediated, at least in part, by a MAPK-dependent mechanism.

432 T-cell anergy and relative sparing of antigen-presenting cells may explain rejection and graft adaptation by regulatory-suppressor cell formation with thymoglobulin in pediatric liver transplantation

432 T-cell anergy and relative sparing of antigen-presenting cells may explain rejection and graft adaptation by regulatory-suppressor cell formation with thymoglobulin in pediatric liver transplantation

Paediatric revision myringoplasty: outcomes and prospects.

Paediatric revision myringoplasty has received little attention. This study addressed the issue exclusively and reviewed the short- and long-term results of surgery in children between the ages of five and 15. Twenty-six out of 38 operated ears (68.4 per cent) were initially intact. The causes of immediate failure in decreasing order were: infection with graft necrosis, complete no-take of the graft and poor anterior adaptation of the graft. Age, size and site of perforation and surgeon's experience did not significantly affect the initial outcome of surgery. Six ears developed delayed re-perforations, thus decreasing the overall sucess rate to 52.6 per cent. The latter were attributable to either episodes of acute otitis media or to insidious atrophy of the tympanic membrane. Notably, none developed post-operative sensorineural hearing loss. It is concluded that the results of paediatric revision myringoplasty are rather disappointing, yet arguments encouraging its practice are favourably presented.

Effect of epidermal growth factor on intestinal adaptation after allogeneic small bowel transplantation in rats

We reported that epidermal growth factor (EGF) stimulated graft adaptation in a rat model of syngeneic small bowel transplantation. However, graft rejection is a severe problem with clinical small bowel transplantation, because small intestinal wall contains large amounts of lymphoid tissue. Studies were performed to investigate the effect of EGF on allogeneic graft adaptation after small bowel transplantation in rats treated with an immunosuppressant FK506. The transplanted animals received intraperitoneally EGF or saline (untreated) after surgery and were examined for analysis one week later. EGF-treated group markedly enhanced the water absorption and induction of sodium glucose cotransporter (SGLT1) as compared with EGF-untreated group. EGF-treated group also increased the mucosal crypt depth and its cell proliferating rate, although there was no significant difference in the mucosal villus height between the two groups. These results indicate that EGF accelerates intestinal allograft adaptation in part by the recovery of mucosal structure and function after small bowel transplantation in rats. EGF may have relevance to promote graft function in clinical small intestinal transplantation.