Intramyocardial implantation of CD133+ stem cells improved cardiac function without bypass surgery.

INTRODUCTION: Cell transplantation for myocardial regeneration has been shown to have beneficial effects on cardiac function after myocardial infarction. Most clinical studies of intramyocardial cell transplantation were performed in combination with coronary artery bypass grafting (CABG). The contribution of implanted stem cells could yet not be clearly distinguished from the effect of the CABG surgery. Our current phase 1 clinical study has focused on the safety and feasibility of CD133+-enriched stem cell transplantation without CABG and its potential beneficial effect on cardiac function. METHOD AND RESULTS: Ten patients with end-stage chronic ischemic cardiomyopathy (ejection fraction <22%) were enrolled in the study. Bone marrow (up to 380 mL) was harvested from the iliac crest. CD133+ cells were purified from bone marrow cells using the CliniMACS device with purities up to 99%. Autologous bone marrow CD133+ cells (1.5-9.7 X 106 cells) were injected into predefined regions. Cardiac functions prior to and 3, 6, and 9 months after cell transplantation were assessed by cardiac magnetic resonance imaging. Stem cell transplantation typically improved the heart function stage from New York Heart Association/Canadian Cardiovascular Society class III-IV to I-II. The mean preoperative and postoperative ventricular ejection fractions were 15.8 +/- 5% and 24.8 +/- 5%, respectively. CONCLUSION: CD133+ injection into ischemic myocardium was feasible and safe. Stem cell transplantation alone improved cardiac function in all patients. This technique might hold promise as an alternative to medical management in patients with severe ischemic heart failure who are ineligible for conventional revascularization.

Autologous transplantation of CD133+ BM-derived stem cells as a therapeutic option for dilatative cardiomyopathy.

We report the case of a 58-year-old man with end-stage non-ischemic cardiomyopathy. Baseline transthoracic echocardiography (TTE) and cardiac magnetic resonance (cMRI) revealed a markedly depressed left ventricle systolic function. He underwent autologous CD133+ BM-derived cell transplantation through a minimally invasive approach. During surgery 19 x 10(6) BM-derived stem cells were injected by the transepimyocardial route. Six months after the operation TTE and cMRI showed a clear improvement in left ventricular contractility.

Autologous bone marrow-derived stem cell therapy in combination with TMLR. A novel therapeutic option for endstage coronary heart disease: report on 2 cases.

We report 2 cases in which patients with coronary heart disease not amenable for conventional revascularization underwent transmyocardial laser revascularization (TMLR) and implantation of AC133+ bone-marrow stem cells. The reason for using TMLR in combination with stem cell application is to take advantage of the synergistic angiogenic effect. The local inflammatory reaction induced by TMLR should serve as an informational platform for stem cells and may trigger their angiogenic differentiation. Functional analysis of myocardial performance after treatment in these 2 cases revealed dramatic improvement of the wall motion at the site of the TMLR and stem cell application. Because TMLR does not enhance myocardial contractility and there was no angiographic evidence of major collaterals to the ischemic region in either patient, we assume that the synergistic effect of stem cells and TMLR-induced angiogenesis occurred; however, our assumption is of a speculative nature. We think that TMLR in combination with stem cell transplantation might become a novel revascularization therapy for ischemic myocardium.

Laminin, hyaluronan, tenascin-C and type VI collagen levels in sera from patients with malignant melanoma.

In this study, hyaluronan, laminin-1, tenascin-C and type VI collagen were measured in the sera of patients with stage I/II and stage IV melanoma. A significant increase in the serum levels of all four extracellular matrix proteins was found in patients with stage IV melanoma compared to healthy donors. Type VI collagen and hyaluronan serum levels were also significantly increased in stage I/II melanoma. Increased expression of the four matrix proteins was also demonstrated in melanoma cell lines using reverse transcriptase- polmerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). We suggest that tenascin-C, hyaluronan, laminin-1 and type VI collagen are involved in melanoma development and extracellular matrix remodelling during melanoma progression. This finding will be of interest in the development of serum markers for progression of malignant melanoma.

Attenuation of CCl(4)-induced hepatic fibrosis by GdCl(3) treatment or dietary glycine.

The role of Kupffer cells in CCl(4)-induced fibrosis was investigated in vivo. Male Wistar rats were treated with phenobarbital and CCl(4) for 9 wk, and a group of rats were injected with the Kupffer cell toxicant gadolinium chloride (GdCl(3)) or were fed glycine, which inactivates Kupffer cells. After CCl(4) alone, the fibrosis score was 3.0 +/- 0.1 and collagen protein and mRNA expression were elevated, but GdCl(3) or glycine blunted these parameters. Glycine did not alter cytochrome P-450 2E1, making it unlikely that glycine affects CCl(4) metabolism. Treatment with GdCl(3) or glycine prevented CCl(4)-induced increases in transforming growth factor (TGF)-beta 1 protein levels and expression. CCl(4) treatment increased alpha-smooth muscle actin staining (score 3.0 +/- 0.2), whereas treatment with GdCl(3) and glycine during CCl(4) exposure blocked this effect (1.2 +/- 0.5); there was no staining with glycine treatment. These results support previous in vitro data and demonstrate that treatment of rats with the selective Kupffer cell toxicant GdCl(3) prevents stellate cell activation and the development of fibrosis.

Autocrine expression of activated transforming growth factor-beta(1) induces apoptosis in normal rat liver.

The aim of this study was to determine the differential effects of latent and activated transforming growth factor (TGF)-beta(1) in growth control of normal and proliferating hepatocytes in vivo. Rats were injected with adenoviruses expressing control transgenes (Ctrl), latent TGF-beta(1) [TGF-beta(L)], or activated TGF-beta(1) [TGF-beta(A)]. Additional animals underwent two-thirds partial hepatectomy (PH) 24 h after injection. Increased hepatocyte apoptosis was observed in TGF-beta(A)-injected but not TGF-beta(L)-injected animals 24 h postinjection (10.5%) compared with Ctrl animals (0.37%). The percent of apoptotic cells increased to 32.1% in TGF-beta(A)-injected animals 48 h after injection. Furthermore, TGF-beta(A)-injected rats did not survive 24 h after PH. Four hours after PH, 0.25 and 14.1% apoptotic hepatocytes were seen in Ctrl- and TGF-beta(A)-injected rats, respectively. TGF-beta(A)-induced apoptosis in primary rat hepatocytes was blocked with a pancaspase inhibitor. Thus autocrine expression of TGF-beta(A) but not TGF-beta(L) induces hepatocyte apoptosis in the normal rat liver. Rats overexpressing TGF-beta(A) do not survive two-thirds PH due to hepatic apoptosis. Thus activation of TGF-beta(1) may be a critical step in the growth control of normal and proliferating rat hepatocytes.

Characterization of the interaction between alphaCP(2) and the 3'-untranslated region of collagen alpha1(I) mRNA.

Activated hepatic stellate cells produce increased type I collagen in hepatic fibrosis. The increase in type I collagen protein results from an increase in mRNA levels that is mainly mediated by increased mRNA stability. Protein-RNA interactions in the 3'-UTR of the collagen alpha1(I) mRNA correlate with stabilization of the mRNA during hepatic stellate cell activation. A component of the binding complex is alphaCP(2). Recombinant alphaCP(2) is sufficient for binding to the 3'-UTR of collagen alpha1(I). To characterize the binding affinity of and specificity for alphaCP(2), we performed electrophoretic mobility shift assays using the poly(C)-rich sequence in the 3'-UTR of collagen alpha1(I) as probe. The binding affinity of alphaCP(2) for the 3'-UTR sequence is approximately 2 nM in vitro and the wild-type 3' sequence binds with high specificity. Furthermore, we demonstrate a system for detecting protein-nucleotide interactions that is suitable for high throughput assays using molecular beacons. Molecular beacons, developed for DNA-DNA hybridization, are oligonucleotides with a fluorophore and quencher brought together by a hairpin sequence. Fluorescence increases when the hairpin is disrupted by binding to an antisense sequence or interaction with a protein. Molecular beacons displayed a similar high affinity for binding to recombinant alphaCP(2) to the wild-type 3' sequence, although the kinetics of binding were slower.

Enzyme immunoassay for the measurement of human tenascin-C on the Bayer Immuno 1 analyzer.

OBJECTIVES: To evaluate a new tenascin-C assay performed on the Bayer Immuno 1 system. DESIGN AND METHODS: The precision was measured using three levels of serum pools. Linearity was tested by diluting patient serum samples containing high tenascin-C concentrations, and the minimal detectable concentration determined by repetitive analysis of the zero calibrator. Preliminary reference intervals were determined by testing serum samples from 220 healthy individuals. Biovariability was estimated in a cohort of 20 apparently healthy subjects over 18 days. The levels of tenascin-C in patients with different liver diseases was tested. RESULTS: The detection limit was 2 ng/mL. At concentrations ranging from 325 to 1957 ng/mL the assay demonstrated within-run and between-run CVs ranging from 4% to 3.6% and 8.4% to 6.7%, respectively. Dilutions of sera were linear and parallel to the standard curve with recoveries ranging from 97% to 100%. The reference interval (central 95% interval) for tenascin-C in serum of healthy adults was 199-906 ng/mL. The variability study yielded an analytical variability, CV(A), of 1.8%; a within-subject variability, CV(I), of 11.7%; and a between-subject variability, CV(G), of 39.3%. Tenascin-C concentrations in sera of liver disease patients were significantly increased. CONCLUSIONS: The novel assay provides a rapid and reliable procedure for the determination of tenascin-C levels in human sera.

Two assays for measuring fibrosis: reverse transcriptase-polymerase chain reaction of collagen alpha(1) (III) mRNA is an early predictor of subsequent collagen deposition while a novel serum N-terminal procollagen (III) propeptide assay reflects manifest fibrosis in carbon tetrachloride-treated rats.

Using a novel quantitative reverse transcriptase-polymerase chain reaction assay, we have determined the amount of specific mRNA for procollagen alpha(1) (III) (PIIIP) in the carbon tetrachloride (CCl(4)) model of liver fibrosis in rats. After a single week of CCl(4) application, the amount of PIIIP mRNA was increased approximately 10 times over the untreated control group and continued to increase to approximately 30 times after 7 weeks of intoxication. In this model substantial fibrosis was demonstrated by computer-aided morphometry after 5 to 7 weeks of treatment. Using recombinant murine N-terminal procollagen alpha(1) (III) propeptide (PIIINP), a novel sensitive immunoassay for the measurement of circulating PIIINP in rodent sera was established. An increase in PIIINP serum levels was observed after 5 to 7 weeks of CCl(4) intoxication. Our results suggest PIIIP gene expression is an early marker of tissue fibrosis. Early PIIIP gene expression is correlated with the extent of the subsequent fibrosis. PIIIP mRNA levels increase much earlier than conventional histological examination or PIIINP levels. PIIINP measurements with our new serum assay, on the other hand, are a good noninvasive marker of manifest fibrosis but are a poor marker of fibrogenesis.

Favorable combination effects of the leukotriene synthesis inhibitor BAY X 1005 and dexamethasone on edema formation in the arachidonic acid-induced mouse ear inflammation test.

The effects of a combination of the leukotriene synthesis inhibitor (LSI) BAY X 1005 with the glucocorticosteroid dexamethasone were studied in the arachidonic acid (AA)-induced mouse ear inflammation test (AA-MEIT). We have determined the dose-dependent effects of dexamethasone to reduce edema formation when a combination of 25 mg/kg BAY X 1005 and increasing dosages of dexamethasone was administered orally (p.o.). The inhibition of ear thicknesses increases with the combination therapy were compared with the inhibition observed when both compounds were applied alone. The edema inhibition at the fixed oral dose of 25 mg/kg p.o. BAY X 1005 was 57+/-2%. Dexamethasone alone dose-dependently inhibited edema formation with a flat inhibition curve at dosages ranging from 0.008 mg/kg (11+/-13%) to 0.5 mg/kg (651+/-11%). In combination with BAY X 1005, the corresponding inhibition curve for dexamethasone was shifted upward starting from 56+/-13% at 0.008 mg/kg. At the two highest dexamethasone dosages (0.125 mg/kg and 0.5 mg/kg) an identical inhibition (86+/-10%) was observed indicating a plateauing of the antiedematous effect of this combination. The results indicate that at suitable dosages (0.031 mg/kg and 0.125 mg/kg) the effects of BAY X 1005 and dexamethasone were additive. To further corroborate the combination effects of BAY X 1005 and dexamethasone the 5-HT receptor antagonist methysergide and the H1 receptor antagonist pyrilamine were employed as a pretreatment to eliminate mouse-specific inflammation responses. In the methysergide/pyrilamine (12.5 mg/kg s.c. each)-conditioned AA-MEIT model 85+/-3% edema reduction were observed with BAY X 1005 and 74+/-3% with dexamethasone. The combination of 25 mg/kg BAY X 1005 and 0.5 mg/kg dexamethasone was slightly more effective in the conditioned AA-MEIT (90+/-3%) than either compound alone. Our results demonstrate that the LSI BAY X 1005 interacted favorably with the glucocorticosteroid dexamethasone suggesting a potentially useful new combination strategy to treat acute inflammatory disease conditions. This effect can be explained on the basis of the mechanisms of action of both therapeutic principles.

The role of TGFbeta1 in initiating hepatic stellate cell activation in vivo.

BACKGROUND/AIMS: The activation of hepatic stellate cells is a key initiating event in hepatic fibrogenesis. Although TGFbeta1 is a potent inducer of collagen alpha1(I) expression in vitro and elevated levels of TGFbeta1 are found in patients and experimental animals with hepatic fibrosis and cirrhosis, the role of increased TGFbeta1 in the initiation of hepatic stellate cell activation in vivo is unknown. We used two experimental approaches to study this relationship: 1) Induction of an acute liver injury with carbon tetrachloride (CCl4) in normal and TGFbeta1-knockout (ko) mice, and 2) overexpression of TGFbeta1 in the liver of wild-type mice using a recombinant replication-deficient adenovirus encoding human TGFbeta1 (Ad-TGFbeta1). METHODS: TGFbeta1-ko mice (n=6) and normal mice (n=6) were injected once intraperitoneally (i.p.) with CCl4 (1 microl/g BW) or mineral oil. Wild-type mice (n=3) were injected intravenously with Ad-TGFbeta1 (10(10) pfu) or a control virus expressing beta-galactosidase (Ad-LacZ, 10(10) pfu). Animals were sacrificed after 3 days and total liver RNA was prepared. The expression of collagen alpha1(I) mRNA normalized to GAPDH mRNA was measured by RNase protection assay, asmooth muscle actin (alpha-sma) protein expression was analyzed by Western blotting. The expression of TGFbeta1, TGFbeta2, and TGFbeta3 mRNAs were determined semi-quantitatively with RT-PCR. RESULTS: The collagen alpha1(I) mRNA was increased 10-fold in CCl4-treated wild-type mice compared to the controls. This increase was reduced about 80% in the TGFbeta1-ko mice. The TGFbeta1 mRNA levels in the wild-type mice were proportional to the collagen alpha1(I) mRNA levels. a-sma, a marker of hepatic stellate cell activation, was expressed earlier and at a higher level in wild-type mice than TGFbeta-ko mice after CCl4 treatment. The Ad-TGFbeta1 infected mice had 14-fold higher hepatic TGFbeta protein levels and 15-fold higher collagen alpha1(I) mRNA levels than the Ad-LacZ-infected control mice. Collagen alpha1(I) mRNA levels were proportional to the transgenic TGFbeta1 mRNA levels, while the endogenous TGFbeta1 was only slightly higher than in the controls. TGFbeta2 and TGFbeta3 mRNA levels were elevated in CCl4-treated wild-type and TGFbeta1-ko mice and in Ad-TGFbeta1-infected mice compared to the controls. CONCLUSIONS: Absence of TGFbeta1 inhibits hepatic collagen alpha1(I) mRNA and alpha-sma protein expression by the toxic stimulus CCl4, and targeted TGFbeta1 overexpression increases collagen alpha1(I) mRNA and alpha-sma protein levels in the liver in vivo. Other TGFbeta family members do not compensate for the TGFbeta1 deficiency. This indicates that TGFbeta1 accelerates, but is not absolutely required, for the activation of hepatic stellate cells.

Epitope-specific monoclonal antibodies against human C-terminal procollagen alpha1(III)-propeptide.

We have generated monoclonal antibodies against recombinant C-terminal human procollagen alpha1(III) propeptide (PIIICP), produced in E. coli in high yields. The monoclonal antibodies were screened for epitope specificity using recombinant truncated PIIICP. Several antibodies were identified which recognized different regions of the PIIICP molecule. The ability of the antibodies to detect PIIICP antigens in human cell line lysates and supernatants was demonstrated. As PIIICP antigens are a key marker of extracellular matrix metabolism, the monoclonal antibodies described here should be of value for clinical and basic research.

Antiedematous effects of combination therapies with the leukotriene synthesis inhibitor BAY X 1005 in the archidonic acid-induced mouse ear inflammation test.

The leukotriene synthesis inhibitor (LSI) BAY X 1005 was tested in the arachidonic acid (AA)-induced mouse ear inflammation test (AA-MEIT) alone and in combination with other representative anti-inflammatory compounds for antiedematous effects. When BAY X 1005 was used as a monotherapy, the ED50 (half-maximal effect) was observed at 5.1 mg/kg per os (p.o.) and at 0.8 microg for topical application. The maximal inhibition of edema formation was estimated to be 63% for p.o. application and 54% for topical application. Furthermore, experiments were carried out in which the animals were conditioned with a combination of the H1/5-HT receptor antagonists pyrilamine and methysergide in addition to treatment with BAY X 1005. This conditioning treatment alone, without BAY X 1005, resulted in a 45 +/- 13% reduction in edema formation. ED50 substance effects were observed at 5.3 mg/kg p.o. and at 0.02 microg per ear for topical application. The maximal inhibition of edema formation in the conditioned groups was 82% for the oral administration of BAY X 1005 and 72% for the topical application. To further characterize the antiinflammatory properties of BAY X 1005 in the conditioned and unconditioned AA-MEIT, BAY X 1005 was tested in combination with the nitric oxide (NO) synthase inhibitor L-NAME, with the cyclooxygenase inhibitor indomethacin, and in combination with both compounds. BAY X 1005 consistently exerted anti-inflammatory effects in the AA-MEIT. The effects of a combination of different inhibitors of inflammatory mediators were not simply additive in this model, as was demonstrated in the case of the combination of L-NAME and indomethacin where a smaller inhibition than with either substance alone was observed. In the conditioned model, a combination of BAY X 1005 with L-NAME or indomethacin, or with both compounds together was less effective than the monotherapy with BAY X 1005. Taken together, these data suggest that cyclooxygenase products and NO have little effect on edema formation in the conditioned and unconditioned AA-MEIT model and that their interaction with leukotrienes is of minor quantitative importance. Our results underline the complexity of the AA-MEIT model and provide a rationale for H1/5-HT-conditioning animals to compensate for peculiarities in the mouse-specific mediator spectrum and to recognize the importance of the leukotriene-specific inflammatory response.

Assessment of G-CSF and GM-CSF mRNA expression in peripheral blood mononuclear cells from patients with severe congenital neutropenia and in human myeloid leukemic cell lines.

Patients with severe congenital neutropenia (SCN), also called Kostmann syndrome, are unable to generate sufficient peripheral blood granulocytes owing to an arrest of myeloid differentiation at the level of promyelocytes. Similarly, myeloid leukemic cells show a maturation arrest at different stages of myeloid maturation coupled with uncontrolled proliferation. Among other cells, defective production of or defective response to granulocyte/macrophage colony-stimulating factor (GM-CSF) or granulocyte CSF (G-CSF) might be involved in the pathophysiology of these disorders of hematopoiesis. Reverse transcription of messenger RNA and subsequent specific amplification by the polymerase chain reaction (RT-PCR) served as a sensitive technique to detect G-CSF and GM-CSF gene expression. We have tested two alternative assays for the specific quantitation of transcript levels for G-CSF. Applying one assay we could demonstrate that: 1) peripheral blood monocytes from 5 patients with SCN are able to express G-CSF and GM-CSF messenger RNA, suggesting that defective production of these factors is not responsible for the neutropenia in this condition; 2) messenger RNA levels from 5 SCN patients were on average higher than the levels determined for three healthy volunteers; 3) 7 of 9 of the examined myeloid cell lines express GM-CSF and all of them G-CSF mRNA. These results show that quantitative PCR techniques can be used as simple tools to elucidate aspects of the pathophysiology of hematologic disorders concerning the production of CSFs.