Analysis of functional renal allograft tolerance with single-dose rapamycin based induction immunosuppression.

The induction of transplantation tolerance is one of the primary goals following solid organ transplantation. The combination of a single dose of rapamycin (RAPA) with a short course of cyclosporine (CsA) has been shown to induce transplantation tolerance in the nonfunctional rat heterotopic cardiac transplant model. The purpose of this study was to assess this effective induction protocol in a functional renal transplant model. Male ACI (RTl(a)) and Lewis (RT1(1)) rats were used as donor and recipients respectively. Allografts received a single RAPA dose of (1.5 mg/kg) combined with CsA (10 mg/kg) 12-14 hr prior to transplantation. CsA (5 mg/kg) was given daily on days +1 - +7. Untreated Lewis to Lewis isografts served as histological controls. Chimerism, assessed in recipient skin, and intragraft interleukin (IL) 10 expression was determined utilizing PCR and RT-PCR techniques respectively. Treated animals and isografts were sacrificed 120-130 days posttransplant for functional and histological evaluation. Allografts (n=9) were functionally tolerant with serum creatinine (0.77+/-0.1 vs. 0.88+/-0.1; P=0.275), blood urea nitrogen (37.6+/-4.6 vs. 23.3+/-1.9; P=0.123), and 24 hr protein excretion (27.0+/-4.4 vs. 17.9+/-5.2; P=0.131) similar to single kidney ACI controls. Histologically, 45% (4/9) allografts were indistinguishable from isografts with no evidence of rejection, and were considered immunologically tolerant. Donor/recipient chimerism was not detected. All immunologically tolerant allografts had evidence of intragraft IL-10 expression. Rejecting allografts and isografts did not express intragraft IL-10. This study confirms the efficacy of pre-engraftment single-dose RAPA combined with CsA in inducing true immunologic tolerance in this stringent functional renal transplant model. The expression of intragraft IL-10 in tolerant recipients suggests a Th-2 shift as the mechanism of tolerance in this model.

Inducible nitric oxide synthase expression in human vein grafts.

BACKGROUND: The patency of vascular reconstructive procedures is limited by the development of intimal hyperplasia (IH). Nitric oxide (NO) seems to be beneficial in abrogating this process. Currently, there is little information concerning inducible nitric oxide synthase (iNOS), the enzyme responsible for NO synthesis, and human vein grafts. The purpose of this study was to examine iNOS gene expression in human aortocoronary vein grafts (ACVG) and infrainguinal vein bypass grafts (IVG). METHODS: Nonthrombosed sections from ACVG (n = 5), IVG (n = 5), and control saphenous vein (SV; n = 4) were harvested and processed for RNA isolation. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed on samples using 32P radioactively end labeled primers. Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was the internal control, and results were expressed as iNOS pmol/GAPDH pmol. RESULTS: There was a significant increase in the iNOS gene expression in the ACVG (0.049 +/- 0.01) when compared with IVG (0.019 +/- 0.001) or normal SV (0.011 +/- 0.002; P < or = 0.05). There was no significant difference between normal vein and the infrainguinal grafts. Sequencing of a fragment of the amplified 428 bp gene product confirmed 84% homology with the available gene bank human sequence. CONCLUSIONS: This study proves that iNOS is expressed in human vein bypass grafts. Additionally, there is a significant elevation of iNOS message in human ACVGs compared with IVG or normal SV. This difference may be the result of the unique vascular beds supplied by these grafts. Ultimately, manipulation of iNOS expression may lead to therapies to alleviate IH in these grafts.

Hypercholesterolemia alters the gene expression of novel components of the extracellular matrix in experimental vein grafts.

The success rate of vascular bypass procedures is limited by the development of intimal hyperplasia (IH). Hypercholesterolemia has been shown to accelerate IH in both arteries and experimental vein grafts; however the mechanism remains uncertain. Hyaluronic acid synthase (HAS-1) is a transmembrane enzyme responsible for the formation of hyaluronan; an important constituent of extracellular matrix (ECM). The integrin receptor for hyaluronan is CD-44. Both CD-44 and HAS-1 have been studied in the development of ECM of wounds but have yet to be examined in the ECM of IH within vein grafts. The purpose of this study was to determine if the expression of CD-44 and HAS-1 is increased during the early stages of IH and how cholesterol supplementation affects these genes. Forty white male New Zealand rabbits were divided into two groups: cholesterol supplemented (1% cholesterol chow) and noncholesterol supplemented. Each set of 20 rabbits was then divided into four additional groups (n = 5); a nonoperative group (control) and three operative groups that underwent a right interposition carotid bypass using jugular vein. Grafts were harvested at 3, 7, and 21 days after surgery for molecular studies and histology. Ribonuclease protection assays were performed using 32P-labeled riboprobes for HAS-1, CD-44, and 18s rRNA. Densitometric analysis is expressed as a ratio (riboprobe/rRNA). Cholesterol levels differed significantly between cholesterol supplemented and nonsupplemented groups (1419 +/- 130 mg/dl and 48 +/- 12 mg/dl) (p < 0.01). There was a significant increase in the expression of HAS-1 and CD-44 in the vein grafts compared to normal jugular vein. Cholesterol supplementation caused a further increase in CD-44 gene expression versus nonsupplemented vein grafts. These data demonstrate a role for CD-44 and HAS-1 transcription in vein graft intimal hyperplasia, which is further altered by cholesterol supplementation. Lastly, these results could explain differences seen in the development of IH with hypercholesterolemia and ultimately provide for improved therapies in alleviating this process.

The nitric oxide precursor L-arginine reduces expression of hyaluronan synthase in experimental vein bypass grafts.

BACKGROUND: The success of vascular bypass procedures is limited by the development of intimal hyperplasia (IH). The nitric oxide (NO) precursor, L-arginine (L-ARG) significantly reduces IH in both arteries and experimental vein grafts; however, the precise mechanism has yet to be elucidated. Hyaluronan synthase-1 (HAS-1) is one of the two enzymes believed to be responsible for making hyaluronan, a key component extracellular matrix composition. PURPOSE: To determine how L-ARG supplementation affects the gene expression of HAS-1 in experimental vein grafts. METHODS: Thirty-four male New Zealand white rabbits were divided into three groups: control (no operation, regular chow and water, n = 4); L-ARG supplemented (n = 15); and no L-ARG (n = 15). The latter two groups underwent a right interposition carotid bypass using jugular vein. Vein grafts were harvested at 7, 14, and 21 days after surgery. Ribonuclease protection assays were performed using 32P-labeled riboprobes for HAS-1 and 18S rRNA as an internal control and expressed as a ratio (HAS-1/rRNA). RESULTS: There was a significant rise in HAS-1 expression in the vein grafts 7 (1.57 +/- 0.5), 14 (0.7 +/- 0.2), and 21 days (2.82 +/- 0.7) after grafting compared to control (0.14 +/- 0.08) (P < 0.05). L-ARG-supplemented animals had a significant decrease in HAS-1 expression at 21 days (0.65 +/- 0.1) compared to nonsupplemented vein grafts (2.82 +/- 0.7) (P < 0.02). CONCLUSIONS: These results demonstrate for the first time a significant rise in HAS expression in the early experimental vein grafts. Furthermore, L-ARG supplementation significantly diminishes the expression of HAS at 21 days. These results may represent a potential mechanism by which augmentation of the L-ARG/NO pathway inhibits IH in experimental vein grafts and may ultimately provide for improved therapeutic interventions in alleviating IH.

The temporal expression of transforming growth factor-beta 1 in early aortocoronary vein grafts.

The success of coronary reconstructive procedures is limited by the high incidence of restenosis secondary to intimal hyperplasia (IH). Transforming growth factor-beta 1 (TGF-beta 1) is a growth factor which has been shown to be important in the early development of IH in arteries and peripheral vein grafts. To date, there is little information concerning the early remodeling in aortocoronary vein grafts (ACVG). The purpose of this study was to characterize the expression of TGF-beta 1 expression in early aortocoronary vein grafts. Eighteen mongrel dogs underwent aortocoronary vein bypass grafting. Vein grafts were excised at 2 hr, 4 hr, and 7 days after implantation, snap frozen, and processed for ribonuclease protection assays (RPA) using 32P-labeled riboprobes for TGF-beta 1 and 18 S rRNA. TGF-beta 1 expression was quantified by densitometric analysis of autoradiographs which were expressed as a ratio TGF-beta 1/rRNA. Representative vessel rings were also collected for histology. There was a significant rise in TGF-beta 1 expression in the 2-hr vein grafts (0.42 +/- 0.04 compared to control saphenous vein (0.21 +/- 0.05, P < 0.02). In addition, there was significant downregulation of TGF-beta 1 at 4 hr (0.28 +/- 0.05) and at 7 days (0.18 +/- 0.01) when compared to 2 hr (P < 0.05). Histological specimens showed minimal intimal hyperplasia at 7 days. These results show for the first time an acute rise in TGF-beta 1 expression in ACVG. This upregulation quickly subsides by 4 hr and gene expression approaches control values by 7 days. By understanding this temporal relationship of expression one could better target potential therapeutic modalities to attenuate IH.

Decreased expression of tissue inhibitor of metalloproteinase 1 in stunned myocardium.

Ultrastructural studies of stunned myocardium have shown disorganization and loss of extracellular collagen and increased collagenase activity early after ischemia and reperfusion. The interplay between matrix metalloproteinase 1 (MMP-1) and tissue inhibitor of metalloproteinase 1 (TIMP-1) regulates the turnover of cardiac extracellular matrix fibrillar collagens. However, the gene expression of MMP-1 and TIMP-1 in stunned myocardium is not known. Here, we determined whether altered expression of MMP-1 and TIMP-1 occurs in globally stunned hearts. An isolated nonworking rabbit heart preparation, perfused with a bovine erythrocyte suspension in modified Krebs solution, was used. Two groups were studied: the stunned group was subjected to 20 min of normothermic global ischemia followed by 120 min of normal reperfusion (n = 8), and the control group underwent 140 min of uninterrupted perfusion (n = 7). The developed pressures at the end of reperfusion for ischemic and control hearts were 67.0 +/- 2.73 and 83.1 +/- 1.52 mm Hg (P < 0. 006) respectively. Ribonuclease protection assays of total left ventricular RNA using riboprobes for MMP-1, TIMP-1, and 18S rRNA were performed. A significant decrease (twofold, P < 0.03) in TIMP-1 gene expression was found in the stunned hearts, while MMP-1 mRNA expression was unchanged. Thus, in early stunning, the decrease in TIMP-1 expression could tip the balance favoring enhanced metalloproteinase activity, promoting collagen turnover, and initiating extracellular matrix remodeling. This may contribute to delayed recovery from myocardial stunning.