Clare R. Gregory

Human tissue-engineered blood vessels for adult arterial revascularization.

There is a crucial need for alternatives to native vein or artery for vascular surgery. The clinical efficacy of synthetic, allogeneic or xenogeneic vessels has been limited by thrombosis, rejection, chronic inflammation and poor mechanical properties. Using adult human fibroblasts extracted from skin biopsies harvested from individuals with advanced cardiovascular disease, we constructed tissue-engineered blood vessels (TEBVs) that serve as arterial bypass grafts in long-term animal models. These TEBVs have mechanical properties similar to human blood vessels, without relying upon synthetic or exogenous scaffolding. The TEBVs are antithrombogenic and mechanically stable for 8 months in vivo. Histological analysis showed complete tissue integration and formation of vasa vasorum. The endothelium was confluent and positive for von Willebrand factor. A smooth muscle–specific α-actin–positive cell population developed within the TEBV, suggesting regeneration of a vascular media. Electron microscopy showed an endothelial basement membrane, elastogenesis and a complex collagen network. These results indicate that a completely biological and clinically relevant TEBV can be assembled exclusively from an individuals own cells.

Treatment with rapamycin and mycophenolic acid reduces arterial intimal thickening produced by mechanical injury and allows endothelial replacement

Rapamycin (RPM) and mycophenolic acid (MPA) inhibit immune responses by antagonizing IL-stimulated lymphocyte activation. These 2 drugs, used alone or preferably in combination, also significantly reduced the response of vascular cells to balloon-catheter arterial injury in rats. When rats were treated for 2 weeks with both drugs starting the day of injury, intimal thickening was significantly reduced (P < 0.001) 14 days after injury; however, by 44 days after injury, intimal thickening had progressed to the extent measured in arteries of untreated control rats. When RPM and MPA were administered for 3 days before and 13 days after injury, arterial intimal thickening was significantly (P = 0.024) reduced and endothelium had regrown in vessels analyzed 44 days after injury. Compared with initiation of treatment on the day of injury, starting the administration of RPM plus MPA before injury appears to limit the activation of cells or actions of factors responsible for the progression of intimal thickening that occurred after the administration of the drugs was terminated. RPM and MPA prevented the development of arterial intimal thickening in a model not dependent upon a rejection response. This direct antiproliferative action on smooth muscle cells by RPM and MPA, in vivo, may prevent the development of arterial intimal thickening associated with chronic rejection.

Rapamycin Inhibits Arterial Intimal Thickening Caused By Both Alloimmune And Mechanical Injury: Its Effect On Cellular, Growth Factor, And Cytokine Responses In Injured Vessels

The effect of rapamycin (RPM) on the extent of arterial intimal thickening was determined in rat recipients of orthotopic femoral artery allografts or in rats that had undergone balloon catheter injury to carotid arteries. In untreated rats, neointima comprised approximately 50% of the arterial wall area in both models. Although treatment of allograft recipients for 40 days with 1.5 mg/kg/day RPM was ineffective, a dose of 6 mg/kg/day (days 0-7) followed by 3 mg/kg/day (days 8-39) reduced intimal thickening by 98% (P < 0.0001). The higher RPM dose reduced T cell and macrophage infiltration significantly and decreased the expression of IL-2 receptor, class II Ag, and mRNAs for growth factors and cytokines. Treatment with 1.5 mg/kg/day RPM (days 0-13) after balloon-catheter injury reduced intimal thickening by 45% (P = 0.0254) and substantially decreased macrophage infiltration and expression of class II Ag in the adventitia. Within the neointima, however, mRNAs for platelet-derived growth factor-alpha, basic fibroblast growth factor, and transforming growth factor-beta were still expressed. In summary, we have shown that RPM inhibits not only the vascular response to injury caused by allograft rejection, but also the response to balloon catheter injury. This new information is important to our understanding of: (1) the fundamental processes responsible for intimal thickening regardless of the cause of vascular injury, (2) mechanisms of action of RPM that explain its effects on the response to very different types of vascular injury, and (3) the potentially diverse therapeutic applications of drugs, like RPM, that inhibit the actions of both immune and nonimmune cytokines and growth factors.

Immunosuppression with a combination of the leflunomide analog, FK778, and microemulsified cyclosporine for renal transplantation in mongrel dogs.

Background. The leflunomide analog, FK778, is a selective pyrimidine synthesis inhibitor. In rodent models, FK778 is efficacious in the prevention of allograft and xenograft rejection, and a combination of FK778 and cyclosporine has synergistic immunosuppressive efficacy. Methods. Heterotopic renal transplantation was performed in 20 dogs. Dogs were randomly assigned to three treatment groups: Neoral alone (n=6), FK778 alone (n=7), or a combination of Neoral and FK778 (n=7). Dogs were killed when the plasma creatinine concentration exceeded 7 mg/dL. A complete postmortem examination was performed and the type of acute–active allograft rejection described. Results. A combination of Neoral and FK778 significantly prolonged allograft survival (P =0.0007), with median survival times of 14.5 days for Neoral alone, 7 days for FK778 alone, and 36 days for Neoral and FK778. Allograft histologic changes were consistent with acute–active allograft rejection in 19 of 20 dogs: in the Neoral-alone group, four dogs were type IB, and two were type IIA; in the FK778-alone group, five dogs were type IB, and one was type IIB; and in the Neoral and FK778 group, three dogs were type IB, three were IIA, and one dog was type III. The dog with type III rejection appeared to experience acute sepsis before rejection. Vomiting, diarrhea, and histologic gastritis and enteritis were commonly observed in dogs treated with the combination of Neoral and FK778. Conclusions. A combination of Neoral and FK778 prolonged allograft survival in a robust rejection model. Further investigation of FK778 in organ transplantation is warranted.

Combination leflunomide and cyclosporine prevents rejection of functional whole limb allografts in the rat

Whole rear limbs were transplanted from Brown Norway or Lewis rat donors to Lewis rat recipients (n=6 per group). One group of allograft recipients was treated with leflunomide (10 mg/kg/24 hr/orally) and cyclosporine (5 mg/kg24 hr/orally) starting 2 days before to surgery. Treatment continued for 60 days or until graft rejection. Untreated allografts were rejected over 6-8 days. After isograft transplantation, weight bearing began by day 17-25 after surgery. Sensory function was restored by 50 days after surgery. All allografts in the drug-treated group survived the 60-day period; survival in this group was significantly longer (P=0.0001) than the untreated controls. Weight bearing began by day 30, but was incomplete in two rats at 60 days. Peroneal nerve function was present in half the rats at 60 days after surgery. Leflunomide combined with cyclosporine prevented whole limb allograft rejection across a major histocompatibility barrier.

The Anti‐Non‐Gal Xenoantibody Response to Xenoantigens on Gal Knockout Pig Cells Is Encoded by a Restricted Number of Germline Progenitors

Antibodies directed at non‐gal xenoantigens are responsible for acute humoral xenograft rejection when gal knockout (GalTKO) pig organs are transplanted into nonhuman primates. We generated IgM and IgG gene libraries using peripheral blood lymphocytes of rhesus monkeys initiating active xenoantibody responses after immunization with GalTKO pig endothelial cells and used these libraries to identify IgVH genes that encode antibody responses to non‐gal pig xenoantigens. Immunoglobulin genes derived from the IGHV3–21 germline progenitor encode xenoantibodies directed at non‐gal xenoantigens. Transduction of GalTKO cells with lentiviral vectors expressing the porcine α1,3 galactosyltransferase gene responsible for gal carbohydrate expression results in a higher level of binding of ‘anti‐non‐gal’ xenoantibodies to transduced GalTKO cells expressing the gal carbohydrate, suggesting that anti‐non‐gal xenoantibodies cross react with carbohydrate xenoantigens. The galactosyltransferase two gene encoding isoglobotriaosylceramide synthase (iGb3 synthase) is not expressed in GalTKO pig cells. Our results demonstrate that anti‐non‐gal xenoantibodies in primates are encoded by IgVH genes that are restricted to IGHV3–21 and bind to an epitope that is structurally related to but distinct from the Gal carbohydrate.

Compared with cyclosporine, ISATX247 significantly prolongs renal-allograft survival in a nonhuman primate model

Background. ISATX247 is a novel calcineurin inhibitor that has shown more potency than cyclosporine in vitro. This is the first study to compare the survival times of renal allografts in nonhuman primates treated with either ISATX247 or cyclosporine. Methods. Adult, male cynomolgus monkeys were divided into blood-group compatible and mixed-lymphocyte, stimulation-mismatched, donor-recipient pairs. Heterotopic renal transplantation and bilateral native nephrectomies were performed. The monkeys were placed into either an ISATX247 or cyclosporine treatment group. Both groups were dosed twice daily to maintain a 12-hour drug-trough level of 150 ng/mL. Whole-blood concentrations of ISATX247 and cyclosporine, complete blood counts, and serum chemistry profiles were performed three times a week. Euthanasia was performed if the serum creatinine concentration became 7 or more mg/dL or a serious complication developed. Results. The group receiving ISATX247 (n=8) survived significantly (P=0.0036) longer than the group receiving cyclosporine (n=7). The mean trough blood concentration of ISATX247 was 120±32 ng/mL and cyclosporine was 189±130 ng/mL. The average area under the curve0–12 for ISATX247 was 6045±1679 ng/mL/hr and for cyclosporine was 4919±823 ng/mL/hr. The average calcineurin inhibition at trough blood concentrations was 80±11% for ISATX247 and 48±12% for cyclosporine. Conclusions. Allografts in monkeys treated with ISATX247 survived significantly longer than those treated with cyclosporine. On the basis of survival times and degree of calcineurin inhibition, ISATX247 is a more potent immunosuppressive agent than cyclosporine in this nonhuman primate model of renal-allograft transplantation.

Effects of leflunomide and cyclosporine on myocutaneous allograft survival in the rat

The immunosuppressive effects of leflunomide and cyclosporine were evaluated in a rat neurovascularized myocutaneous allograft model. Inbred Brown-Norway and Lewis rats were served as donors and recipients, respectively. All recipients were observed for 60 days or until allograft rejection occurred. All isograft controls (Lewis to Lewis, n=6) survived uneventfully. All control allografts (n=6) were rejected within 6 days. Allograft recipients (n=6) administered leflunomide (10 mg/kg/24 hr) rejected their allografts in 28.50+/-6.12 days, and allograft recipients (n=6), administered cyclosporine (5 mg/kg/24 hr) rejected their allografts in 24.33+/-10.48 days. When allograft recipients were administered a combination of leflunomide and cyclosporine (10 mg/kg/24 hr and 5 mg/kg/24 hr, respectively), all allografts survived to 60 days with only partial rejection of the skin of one graft. The neuromuscular function of the allografts of the rats receiving combination therapy was comparable to that of the isografts. The combination of leflunomide and cyclosporine controlled myocutaneous allorejection despite a strong immunological challenge.

Cyclosporine pharmacokinetics in cats following topical ocular administration

Topical ocular administration of two forms of cyclosporine were studied in the cat. Both forms were able to produce measurable whole-blood levels capable of suppressing in vitro lymphocyte stimulation. The kinetics of cyclosporine following administration of either oral solution or cyclosporine in olive oil were variable, with peak concentrations ranging from 450 to 1033 ng/ml and 288 to 648 ng/ml, respectively. Absorption lag time ranged from 0 to 1.34 hr for oral solution, and 0.27 to 1.2 hr for cyclosporine in olive oil. The half-life of elimination ranged from 2.41 to 10.04 hr, and 3.09 to 15.75 hr, respectively. When compared with the commercially available oral solution, cyclosporine dissolved in olive oil was better tolerated during administration. Topical ocular administration of cyclosporine in cats offers a possible alternative method of treatment for individuals intolerant of oral administration. Topical ocular administration might also replace the need for intravenous administration of cyclosporine during perioperative periods or during periods of vomiting and nausea associated with rejection or other illnesses. Due to individual variation in absorption and elimination of topically applied cyclosporine, dosages in each cat must be determined by monitoring blood, plasma, or serum levels.

Fluvastatin in combination with RAD significantly reduces graft vascular disease in rat cardiac allografts

Background. RAD is a potent immunosuppressive agent that has been shown to be effective in preventing acute and chronic allograft rejection in animal models. The HMGCoA reductase inhibitors have been found to reduce the incidence of graft vascular disease (GVD) in heart transplant patients and in animal models. This study was designed to investigate the effects of fluvastatin or pravastatin in a rodent model of GVD produced using low doses of RAD to prevent acute rejection. Methods. Hearts from Fisher 344 rats were heterotopically transplanted to Lewis rat recipients. RAD was administered orally at 0.5 mg/kg per day for days 0 to 14 and then 0.25 mg/kg per day for an additional 85 days to prevent acute rejection but allow for the development of GVD. Pravastatin (20 mg/kg per day) or fluvastatin (2 or 6 mg/kg per day) was added to the RAD treatment. At the end of a 100-day treatment period, the hearts were harvested for morphometric and histopathologic examinations. Results. Rats treated with fluvastatin, at either dose, had a significant (P ≤0.0239) decrease in coronary arterial intimal thickening (GVD) of approximately 43%. Rats treated with pravastatin had a 22% reduction in GVD that did not reach statistical significance. Treatment with fluvastatin, but not pravastatin, decreased the degree of endomyocardial mononuclear cell infiltration seen with RAD administered alone. Conclusions. Fluvastatin significantly decreased GVD in a rat model produced using low-dose RAD immunosuppression. To a lesser extent, pravastatin also decreased GVD in this model. These data lend further support for the study of fluvastatin, pravastatin, and other HMG-CoA reductase inhibitors for the prevention of GVD in cardiac transplant patients.