Ischemia-Reperfusion Injury Enhances Lymphatic Endothelial VEGFR3 and Rejection in Cardiac Allografts.

Organ damage and innate immunity during heart transplantation may evoke adaptive immunity with serious consequences. Because lymphatic vessels bridge innate and adaptive immunity, they are critical in immune surveillance; however, their role in ischemia-reperfusion injury (IRI) in allotransplantation remains unknown. We investigated whether the lymphangiogenic VEGF-C/VEGFR3 pathway during cardiac allograft IRI regulates organ damage and subsequent interplay between innate and adaptive immunity. We found that cardiac allograft IRI, within hours, increased graft VEGF-C expression and lymphatic vessel activation in the form of increased lymphatic VEGFR3 and adhesion protein expression. Pharmacological VEGF-C/VEGFR3 stimulation resulted in early lymphatic activation and later increase in allograft inflammation. In contrast, pharmacological VEGF-C/VEGFR3 inhibition during cardiac allograft IRI decreased early lymphatic vessel activation with subsequent dampening of acute and chronic rejection. Genetic deletion of VEGFR3 specifically in the lymphatics of the transplanted heart recapitulated the survival effect achieved by pharmacological VEGF-C/VEGFR3 inhibition. Our results suggest that tissue damage rapidly changes lymphatic vessel phenotype, which, in turn, may shape the interplay of innate and adaptive immunity. Importantly, VEGF-C/VEGFR3 inhibition during solid organ transplant IRI could be used as lymphatic-targeted immunomodulatory therapy to prevent acute and chronic rejection.

Donor Heart Treatment With COMP-Ang1 Limits Ischemia-Reperfusion Injury and Rejection of Cardiac Allografts.

The major cause of death during the first year after heart transplantation is primary graft dysfunction due to preservation and ischemia-reperfusion injury (IRI). Angiopoietin-1 is a Tie2 receptor-binding paracrine growth factor with anti-inflammatory properties and indispensable roles in vascular development and stability. We used a stable variant of angiopoietin-1 (COMP-Ang1) to test whether ex vivo intracoronary treatment with a single dose of COMP-Ang1 in donor Dark Agouti rat heart subjected to 4-h cold ischemia would prevent microvascular dysfunction and inflammatory responses in the fully allogeneic recipient Wistar Furth rat. COMP-Ang1 reduced endothelial cell-cell junction disruption of the donor heart in transmission electron microscopy during 4-h cold ischemia, improved myocardial reflow, and reduced microvascular leakage and cardiomyocyte injury of transplanted allografts during IRI. Concurrently, the treatment reduced expression of danger signals, dendritic cell maturation markers, endothelial cell adhesion molecule VCAM-1 and RhoA/Rho-associated protein kinase activation and the influx of macrophages and neutrophils. Furthermore, COMP-Ang1 treatment provided sustained anti-inflammatory effects during acute rejection and prevented the development of cardiac fibrosis and allograft vasculopathy. These results suggest donor heart treatment with COMP-Ang1 having important clinical implications in the prevention of primary and subsequent long-term injury and dysfunction in cardiac allografts.

Angiopoietin-2 inhibition prevents transplant ischemia-reperfusion injury and chronic rejection in rat cardiac allografts.

Transplant ischemia-reperfusion injury (Tx-IRI) and allograft dysfunction remain as two of the major clinical challenges after heart transplantation. We investigated the role of angiopoietin-2 (Ang2) in Tx-IRI and rejection using fully MHC-mismatched rat cardiac allografts. We report that plasma levels of Ang2 were significantly enhanced in the human and rat recipients of cardiac allografts, but not in the rat recipients of syngrafts, during IRI. Ex vivo intracoronary treatment of rat cardiac allografts with anti-Ang2 antibody before 4-h cold preservation prevented microvascular dysfunction, endothelial cell (EC) adhesion molecule expression and leukocyte infiltration, myocardial injury and the development of cardiac fibrosis and allograft vasculopathy. Recipient preoperative and postoperative treatment with anti-Ang2 antibody produced otherwise similar effects without effecting microvascular dysfunction, and in additional experiments prolonged allograft survival. Recipient preoperative treatment alone failed to produce these effects. Moreover, ex vivo intracoronary treatment of allografts with recombinant Ang2 enhanced Tx-IRI and, in an add-back experiment, abolished the beneficial effect of the antibody. We demonstrate that neutralization of Ang2 prevents EC activation, leukocyte infiltration, Tx-IRI and the development of chronic rejection in rat cardiac allografts. Our results suggest that blocking Ang2 pathway is a novel, clinically feasible, T cell-independent strategy to protect cardiac allografts.

Differential effects of pharmacological HIF preconditioning of donors versus recipients in rat cardiac allografts.

Ischemia-reperfusion injury (IRI) induces hypoxia-inducible factor-1 (HIF-1) in the myocardium, but the consequences remain elusive. We investigated HIF-1 activation during cold and warm ischemia and IRI in rat hearts and cardiac syngrafts. We also tested the effect of HIF-α stabilizing prolyl hydroxylase inhibitor (FG-4497) on IRI or allograft survival. Ex vivo ischemia of the heart increased HIF-1α expression in a time- and temperature-dependent fashion. Immunohistochemistry localized HIF-1α to all cardiac cell types. After reperfusion, HIF-1α immunoreactivity persisted in smooth muscle cells and cardiomyocytes in the areas with IRI. This was accompanied with a transient induction of protective HIF-1 downstream genes. Donor FG-4497 pretreatment for 4 h enhanced IRI in cardiac allografts as evidenced by an increase in cardiac troponin T release, cardiomyocyte apoptosis, and activation of innate immunity. Recipient FG-4497 pretreatment for 4 h decreased infiltration of ED1(+) macrophages, and mildly improved the long-term allograft survival. In syngrafts donor FG-4497 pretreatment increased activation of innate immunity, but did not induce myocardial damage. We conclude that the HIF-1 pathway is activated in heart transplants. We suggest that pharmacological HIF-α preconditioning of cardiac allografts donors would not lead to clinical benefit, while in recipients it may result in antiinflammatory effects and prolonged allograft survival.

Association between dental health and acute myocardial infarction.

Known risk factors for coronary heart disease do not explain all of the clinical and epidemiological features of the disease. To examine the role of chronic bacterial infections as risk factors for the disease the association between poor dental health and acute myocardial infarction was investigated in two separate case-control studies of a total of 100 patients with acute myocardial infarction and 102 controls selected from the community at random. Dental health was graded by using two indexes, one of which was assessed blind. Based on these indexes dental health was significantly worse in patients with acute myocardial infarction than in controls. The association remained valid after adjustment for age, social class, smoking, serum lipid concentrations, and the presence of diabetes. Further prospective studies are required in different populations to confirm the association and to elucidate its nature.

Association of hue discrimination loss and diabetic retinopathy.

The results of Farnsworth-Munsell 100-hue, visual acuity, and visual field testing were compared with the severity of retinopathy in a group of 90 diabetic patients. The patients showed significantly higher than expected Farnsworth-Munsell 100-hue scores, with a tritanlike axis, compared with published age norms for nondiabetic individuals. The magnitude of the acquired blue-yellow hue discrimination defect correlated significantly and to a similar extent with both the severity of overall diabetic retinopathy and the severity of macular edema and hard exudate formation. Visual acuity loss correlated somewhat more significantly with macular edema than with overall retinopathy, whereas the converse was true for visual fields. For all visual function tests, the correlations were more significant for fluorescein leakage in the macula than for capillary nonperfusion in the macula. Abnormal hue discrimination was found in 65% (32/49) of eyes with proliferative diabetic retinopathy, suggesting a potential role for this test in screening for proliferative diabetic retinopathy in primary care facilities. Also, because the ability of diabetic patients with color vision deficiency to perform color-dependent tests for urinary and blood glucose may be impaired, such patients should be made aware of this potential problem.

Reduced incidence of rat colon cancer metastases by perioperative immunostimulation with maleic anhydride-divinyl ether-2 (MVE-2).

Intra-abdominal operations result in profound immunodepression during a period when tumor cells are released into the systemic and portal circulations. This combination may augment tumor metastases. The authors have developed a model in which rat colon carcinoma cells transplanted into the portal vein consistently induce hepatic metastases by four weeks, and death within nine weeks. Additionally, the authors have shown that perioperative treatment with levamisole significantly reduces the incidence of metastases. This study tested whether maleic anhydride-divinyl ether-2 (MVE-2), a known immunostimulant, would produce similar effects. Rats pretreated with MVE-2 the day before and day of tumor implantation developed fewer metastases (34 percent of animals treated with MVE-2, compared with 5 percent of animals not treated with MVE-2 had less than or equal to two liver metastases). Eighteen percent of MVE-2-treated rats developed no hepatic metastases. Comparison of median liver weights between the MVE-2-treated group and the nontreated, tumor-bearing group was significant (P = 0.03) and the MVE-2-treated animals had significantly prolonged survival (P = 0.04). The authors conclude that the perioperative period is critical for the implantation and growth of metastases and that perioperative immunostimulation may be a factor in decreasing the incidence of metastases. This model may have relevance to the adjuvant treatment of human colonic cancer.

Abnormalities of the foveal avascular zone in diabetic retinopathy.

The dimensions of the foveal avascular zone (FAZ) were measured in fluorescein angiograms from 36 diabetic patients and 20 nondiabetic controls. The median values for longest diameter, mean diameter, and circumference were significantly greater in the diabetic group than in the control group. Longest diameters greater than 1.0 mm were found almost exclusively in eyes with proliferative diabetic retinopathy. The FAZ dimensions were strongly positively correlated with the severity of capillary nonperfusion in the posterior retina, but not with fluorescein leakage. The presence of proliferative diabetic retinopathy was also strongly correlated with capillary nonperfusion. Retinal capillary occlusion as the cause of FAZ enlargement in diabetic retinopathy is supported by these findings.

Prevention of rat colon cancer metastases by perioperative immunostimulation.

Major intra-abdominal operations result in profound immunodepression. In addition, manipulation of malignant tumors may release tumor cells into the systemic and portal circulations. The additive effects of immunodepression and tumor cell release may enhance the metastatic potential of tumors. Perioperative correction of immune depression by levamisole can restore lymphocyte proliferation levels in rats. We have developed a model in which rat colon carcinoma cells transplanted into the portal venous system consistently induce hepatic metastases by 4 weeks and death within 9 weeks. Rats pretreated with levamisole (4 mg/kg administered intraperitoneally) the day before and the day of tumor implantation developed fewer metastases (41% of animals treated with levamisole compared with 6% of animals not treated with levamisole had less than or equal to two metastases per liver). Twenty percent of the rats treated with levamisole developed no hepatic metastases. Comparison of median liver weights between the group treated with levamisole and the nontreated, tumor-bearing group was highly significant (p less than 0.005). We conclude that the perioperative period is critical for the implantation and growth of metastases and that perioperative immunostimulation may be a factor in decreasing the incidence of metastases. This model may have relevance to the adjuvant treatment of human colon cancer.

Diabetic retinopathy. Assessment of severity and progression.

This study describes a classification of overall retinopathy severity based on the presence and severity of lesions graded in the Airlie House classification of diabetic retinopathy, and demonstrates its use in assessing progression of retinopathy over a 6-year period in a group of insulin-taking patients. One hundred and ninety-one insulin-taking patients with diabetes of at least 5 years' duration were identified from their doctors' records in 1970-1971. Patients were seen again in 1972-1973 and 1976-1977. Stereoscopic color fundus photographs of 7 fields in each eye were taken at each visit. All photos were graded using a modification of the Airlie House classification, which specified 6 levels of retinopathy for a given eye; when both eyes are considered an 11-step grading scheme results. Of all patients seen at the 2-year visit whose retinopathy was at risk of progressing, 41.2% showed progression of one level or more on the scale, 19.2% progression of two or more levels. At the 6-year examination comparable rates were 75.0% and 58.4%. Of patients whose retinopathy was level 4 in each eye (nonproliferative retinopathy of "moderate" severity) at the baseline visit, 13/21 (72%) had progressed to proliferative retinopathy in at least one eye at the 6-year examination. The classification scheme proposed appears useful for characterizing overall retinopathy severity of patients on the basis of gradings of fundus photographs. The data presented may be of help in planning trials of treatment aimed at slowing the development or progression of retinopathy.

Tumor specific reactivity during development of N-methyl-N-nitrosourea-induced rat colon cancer.

Invasive colon adenocarcinomas with lymph node metastases can be induced in Sprague-Dawley rats by 15 weekly intrarectal injections of 2 mg N-methyl-N-nitrosourea (NMU). Extracts were prepared from invasive adenocarcinomas and normal rat colon mucosa by a 2 phase gradient. Mixed leukocyte tumor interaction (MLTI) assays stimulating lymphocytes from tumor-bearing and normal rats were performed using these extracts. Quadruplicate cultures were established with 2 X 10(5) lymphocytes and tumor or normal colon extract. Cultures were pulsed with H3 thymidine at 7 days and harvested 6 hours later. Results were expressed as net counts (experimental CPM minus background CPM). Reactivity in tumor-bearing animals was first seen when rat colons showed intraepithelial dysplasia histologically and was maximal when early invasive colon tumors were present. No difference in stimulation was seen between tumor-bearing and normal animal lymphocyte reactivity with normal colon extract. In conclusion, animals with NMU-induced rat colon cancer show specific tumor reactivity in MLTI assays. Immune reactivity in these animals may provide clues to clinical tumor status by immunologic assay.