Reversal of metabolic block in glycolysis by enzyme replacement in triosephosphate isomerase-deficient cells.

Inherited deficiency of the housekeeping enzyme triosephosphate isomerase (TPI) is the most severe clinical disorder of glycolysis. Homozygotes manifest congenital hemolytic anemia and progressive neuromuscular impairment, which in most cases pursues an inexorable course with fatal outcome in early childhood. No effective therapy is available. Hitherto specific enzyme replacement has not been attempted in disorders of glycolysis. Primary skeletal muscle myoblasts and Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines generated from homozygous TPI-deficient patients were cultured in the presence of exogenous enzyme or cocultured with human K562 erythroleukemia cells as an exogenous source of TPI. Uptake of active enzyme by TPI-deficient cells resulted in reversal of intracellular substrate accumulation, with a reduction in dihydroxyacetone phosphate (DHAP) concentration to levels seen in TPI-competent cells. Evidence of successful metabolic correction of TPI deficiency in vitro establishes the feasibility of enzyme replacement therapy, and has important implications for the potential role of allogeneic bone marrow transplantation and gene therapy as a means of sustained delivery of functional enzyme in vivo.

The consequence of nucleotide substitutions in the triosephosphate isomerase (TPI) gene promoter.

Mutations at -5A-->G, -8-->GA within the cap proximal element (CPE), and -24T-->G within the TATA box of the triosephosphate isomerase (TPI) gene promoter have been identified in populations with a wide geographical distribution. These mutations lie within, or in close proximity to, known cis-active elements in the TPI gene promoter. To determine the functional significance of mutation at these sites, which remains controversial, their effect on the expression of erythrocyte TPI enzyme activity was studied in 110 healthy unrelated subjects. The -5G mutation did not alter erythrocyte TPI level, whereas the -8A mutation was accompanied by a significant reduction in enzyme activity to around 90% and 76% of normal erythrocyte TPI activity in heterozygotes and homozygotes, respectively. The -8A -24G genotype was associated with 75% of normal TPI activity in a heterozygote studied, implying that substitution of G at position -24 within the canonical TATA motif causes an additive decrease in TPI gene transcription in erythroid cells. A DNA-protein complex of 125kDa which was competitively blocked by specific unlabelled oligomers was demonstrated at the CPE and TATA box by electrophoretic mobility shift analysis. These findings provide direct evidence that TPI promoter mutations are linked to a reduction of TPI enzyme activity in vivo.

Ancestral origin of variation in the triosephosphate isomerase gene promoter.

A high frequency of nucleotide substitutions -5A/G, -8G/A, -24T/G in the triosephosphate isomerase (TPI) gene promoter has been demonstrated in African-Americans. The biological significance of these promoter variants, two of which, -8G/A and -24T/G, occur within regulatory elements essential for transcription, is controversial. The geographical distribution and frequency of allelic variation in the TPI promoter was determined in 378 unrelated normal subjects from sub-Saharan African (n = 103), Caribbean (n = 26), Northern European (n = 57), Mediterranean (n = 55), Middle Eastern (n = 42), Asian Indian (n = 48) and Oriental (n = 47) populations. Five haplotypes were identified: the common haplotype, -5A-8G-24T, -5G, -8A, -5G-8A, and -5G-8A-24G. All, with the exception of the -8A haplotype, were present in geographically dispersed populations. The -5G allele, which was found at varying frequency in all groups, has attained high frequency in the African, Caribbean and Oriental populations. Phylogenetic comparison suggests this may represent the ancestral promoter haplotype. Homozygosity for the -5G-8A haplotype identified in four subjects confirms that these variants are not responsible for a null allele as formerly postulated. Linkage disequilibrium between related TPI promoter haplotypes, -5G, -5G-8A and -5G-8A-24G, and a single nucleotide polymorphism at nt2262 of the TPI gene supports a single ancestral origin for these mutations which precedes the separation of African and non-African populations.

Ancestral origin of variation in the triosephosphate isomerase gene promoter

A high frequency of nucleotide substitutions -5A/G, -8G/A, -24T/G in the triosephosphate isomerase (TPI) gene promoter has been demonstrated in African-Americans. The biological significance of these promoter variants, two of which, -8G/A and -24T/G, occur within regulatory elements essential for transcription, is controversial. The geographical distribution and frequency of allelic variation in the TPI promoter was determined in 378 unrelated normal subjects from Sub-Saharan African (n = 103), Caribbean (n = 26), Northern European (n = 57), Mediterranean (n = 55), Middle Eastern (n = 42), Asian Indian (n = 48) and Oriental (n = 47) populations. Five haplotypes were identified: the common haplotype, -5A-8G-24T, -5G, -8A, -5G-8A, and -5G-8A-24G. All, with the exception of the -8A haplotype, were present in geographically dispersed populations. The -5G allele, which was found at varying frequency in the African, Caribbean and Oriental populations. Phylogenetic comparison suggests this may represent the ancestral promoter haplotype. Homozygosity for the -5G-8A haplotype identified in four subjects confirms that these variants are not responsible for a null allele as formerly postulated. Linkage disequilibrium between related TPI promoter haplotypes, -5G, -5G-8A and -5G-8A-24G, and a single nucleotide polymorphism at nt2262 of the TPI gene supports a single ancestral origin for these mutations which preceeds the separation of African populations.(Au)

Regulation of triosephosphate isomerase (TPI) gene expression in TPI deficient lymphoblastoid cells.

The metabolic defect of triosephosphate isomerase (TPI) deficiency is reversible in deficient lymphoblastoid cells when cultured in the presence of human K562 erythroleukemia cells or plasma as exogenous source of functional enzyme. However, plasma contains a variety of undefined biological response modifiers whose effects on TPI gene expression are unknown. In the present study, TPI deficient lymphoblastoid cells were cultured in serum-free medium for 24 h (controls) and stimulated with fresh frozen plasma (FFP) at final concentrations of 20, 40, and 60% for 9 h. Changes in TPI mRNA expression were monitored by slot and Northern blot hybridisations using a specific human TPI cDNA probe. For equivalent loading of total RNA, TPI mRNA expression in FFP-treated lymphoblastoid cells exceeded that for controls by on average 20-fold. Additional studies with the transcription inhibitor, actinomycin D, revealed a rapid degradation of TPI mRNA in controls compared to FFP-treated cells, indicating that the stability of the TPI transcript was affected by plasma. These data suggest that functional or regulatory elements within the TPI gene promoter can be modulated by biological response modifiers. An understanding of the transcriptional control of TPI may provide useful insights into the development of gene therapy strategies for TPI deficiency.

The feasibility of replacement therapy for inherited disorder of glycolysis: triosephosphate isomerase deficiency (review).

Triosephosphate isomerase (TPI, EC 5.3.1.1) is an ubiquitously expressed enzyme that catalyses the interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate in the energy-generating glycolytic pathway. Inherited defects in the TPI gene are characterised biochemically by markedly reduced TPI enzyme activity in all tissues resulting in metabolic block in glycolysis, with accumulating DHAP particularly in red cells. Clinical TPI deficiency is a rare autosomal recessive multi-system disorder characterised by non-spherocytic haemolytic anaemia, recurrent infections, cardiomyopathy, severe and fatal neuromuscular dysfunctions. Reviews of current literature show that after 30 years since TPI deficiency was first described, the disease still remains without effective therapy. However, several potential therapeutic strategies exist for the treatment of inherited metabolic disorders such as TPI deficiency. Development of an effective therapy for TPI deficiency presents a fascinating and formidable challenge for basic laboratory and clinical research. The major aim of this overview is to discuss the current knowledge of TPI deficiency with special emphasis on research efforts directed towards reversing the metabolic effects of the disorder.

The role of natriuretic peptides in cardiovascular disorders and human cardiac allografts (Review).

The human myocardium is composed of a variety of cardiac cell types that synthesise cardioactive factors with autocrine, paracrine and endocrine functions. These cardioactive factors include a family of structurally and functionally related peptides, natriuretic peptides (NPs), that act as cardiovascular cell growth modulatory factors and have significant influence on the regulation of cardiovascular function. The three members of the NP family are atrial natriuretic peptide, brain natriuretic peptide and C-type natriuretic peptide. NPs are ubiquitously expressed in cardiovascular tissues and have specific receptors in cardiac cells, brain cells, vascular endothelial cells and extracardiac tissues through which they elicit a variety of biological responses including natriuresis, diuresis and vasodilation. Cardiac transplantation is the most effective and definitive treatment for end-stage cardiac failure in humans. Orthotopic cardiac transplantation and cardiovascular disorders including hypertension, cardiac hypertrophy, cardiac failure, result in increased myocardial expression of NPs, suggesting a pathophysiological role for these peptides in the heart. The mechanism by which the expression of NPs is regulated in the transplanted human heart is not well understood. Understanding the mechanism of local expression of NPs and their interactions with other putative growth regulatory factors in the human heart may have important implications for potential management of cardiovascular disorders and cardiac transplantation. This article will discuss the current knowledge of NPs in cardiovascular disorders. Most of the emphasis will focus on their possible role in human cardiac allografts as there have been no reviews on this important topic.

Identification of endothelial antigens relevant to transplant coronary artery disease from a human endothelial cell cDNA expression library.

Accelerated transplant coronary artery disease (TxCAD) results in increased expression of antiendothelial antibodies whose target antigens remain largely unidentified. One of these endothelial antigens has been identified as vimentin, a cytoskeletal protein present in cells of the blood vessel walls. In the present study, SDS-PAGE and Western blot analysis of human endothelial cell (EAHy 926) lysates probed with sera from a TxCAD patient were used to confirm immunoreactivity of antiendothelial antibodies towards several endothelial proteins. To further elucidate the identity of these putative antigens, a human endothelial cell (EAHy 926) cDNA expression library was immunoscreened with serum obtained from a TxCAD patient. Two positive cDNA clones were identified by partial nucleotide sequence analysis and GenBank/EMBL database searches for homology as the 85 kDa human CD36 antigen (a cell surface glycoprotein expressed in various cells including epithelial and endothelial cells) and a 50 kDa keratin-like protein (a member of the intermediate filament protein expressed in epithelial cells). These results are the first to demonstrate that human CD36 antigen and a keratin-like protein may be additional target proteins for the anti-endothelial antibodies associated with TxCAD.

Measurement of circulating immunoreactive alpha-atrial natriuretic peptide in human plasma: validation and clinical application.

Recent evidence suggests that plasma alpha-atrial natriuretic peptide (alpha-ANP) may serve as a useful biochemical marker of severe heart failure, as circulating levels become ighly elevated. In the present study, a specific radioimmunoassay (RIA) protocol was adapted and optimised for rapid quantitative measurement of circulating alpha-ANP levels in plasma taken from clinically normal subjects and heart transplant recipients with no evidence of heart failure or heart transplant rejection. The plasma concentration of immunoreactive alpha-ANP in heart transplant recipients (115 +/- 10 pg/ml, n = 14) was significantly higher (p < 0.01) than in normal subjects (14 +/- 5 pg/ml, n = 20). Comparison of circulating plasma immunoreactive alpha-ANP levels obtained by the adapted assay to levels obtained by a standard RIA protocol revealed a significant correlation (r = 0.998, y = 1.004 x -2.94, n = 40). The adapted assay is simple, precise, and with a shorter incubation time that would enable results to be available within one working day. These results show that circulating biological active a-ANP levels are elevated in healthy heart transplant recipients with no evidence of heart failure; thus extending previous reports concerning the elevation in circulating alpha-ANP levels from patients with a wide range of heart disorders. The adapted RIA protocol would facilitate rapid detection and quantification of alpha-ANP in experimental research and routine clinical investigations.

The role of anti-endothelial antibodies in the immunopathogenesis of transplant associated coronary artery disease (Review).

Transplant coronary artery disease (TxCAD) is manifest as a diffuse, concentric intimal proliferation which results in occlusion of the allograft vessel lumen, and is responsible for limiting the long-term success of cardiac transplantation. The recent discovery of high circulating levels of anti-endothelial antibodies (AEAs) in patients with TxCAD has resulted in increased clinical and experimental research interests in understanding their patho-physiological roles in TxCAD. Increasing evidence suggests that AEAs are cross-reactive towards an endothelial protein doublet of 56-58 kDa which has now been characterised and identified as the cytoskeletal protein vimentin. Despite this recent progress the immunopathogenesis of TxCAD remains unclear. In this review recent developments and mechanisms of the involvement of AEAs in the immunopathogenesis of TxCAD are discussed.

Molecular cloning and expression of 56-58 KD antigen associated with transplant coronary artery disease.

High circulating levels of anti-endothelial antibodies have been reported in patients with accelerated transplant coronary artery disease (TxCAD); however, the nature or characteristics of the antigen to which these antibodies bind in vivo remain unclear. To identify the antigen, a human endothelial cell cDNA expression library was constructed in lambdaZAP and immuno-screened with serum containing a high titre of anti-endothelial antibodies. Nucleotide sequence of cloned cDNA inserts and GenBank database searches revealed a 94% identity in a 35 bp overlap to the human vimentin gene. The cloned antigen fusion protein co-migrated with human vimentin of molecular mass 56-58 KD in SDS-PAGE and exhibited immunoreactivity towards monoclonal and polyclonal anti-human vimentin antibodies. These results suggest that the 56-58 KD antigen associated with accelerated TxCAD is vimentin (or a vimentin-like protein), a cytoskeletal protein present in cells of the blood vessel walls.

Metabolic correction of triose phosphate isomerase deficiency in vitro by complementation.

Inherited deficiency of triose phosphate isomerase (TPI), the enzyme that catalyses the interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate, is characterised by an accumulation of intracellular DHAP and markedly reduced enzyme activity in cells and tissues, resulting in progressive, usually fatal neuromuscular dysfunction. Since specific enzyme replacement for TPI deficiency is not currently available, the secretion and recapture of the missing enzyme was investigated in a co-culture model comprising K562 human erythroleukaemia cells and lymphoblastoid cells taken from a TPI deficient patient. A sevenfold reduction in intracellular DHAP with concomitant increase in intracellular TPI activity from 7.25 +/- 0.1 to 197.2 +/- 10 units/mg protein was achieved for co-cultured lymphoblastoid cells. These novel results confirm the existence of a transport mechanism which permits transfer of active TPI from K562 cells to deficient cells, and may have important implications for developing different therapeutic approaches for TPI deficiency and other metabolic disorders of glycolysis.

Insulin-like growth factor I (IGF-I) receptor/binding protein in human diabetic epiretinal membranes.

Insulin-like growth factor I (IGF-I) is thought to play a role in the development of proliferative diabetic retinopathy. Proliferative diabetic retinopathy is characterised by the formation of fibrovascular epiretinal membranes, and IGF-I may initiate and/or potentiate this epiretinal proliferation. To evaluate further the part played by IGF-I in the development of epiretinal tissue, we investigated the presence of IGF-I receptor/binding protein in proliferating diabetic fibrovascular epiretinal membranes. Five fibrovascular epiretinal membranes were obtained by vitrectomy from five patients with proliferating diabetic retinopathy. The presence of IGF-I receptors was investigated by autoradiography using 125I-labeled IGF-I on frozen sections. To characterise binding specificity, some sections were preincubated with either insulin or unlabeled IGF-I. Sections of post-mortem liver were used as controls. Strong labeling of cells with 125I-labeled IGF-I was observed in all epiretinal membranes and in liver cells. Almost no autoradiographic labeling was observed in sections that had been blocked with non-radioactive IGF-I, and very little labeling was found following blockage with insulin. Our preliminary study suggests the presence of IGF-I receptor/binding protein in human diabetic epiretinal membranes. These results support the hypothesis that IGF-I may be involved in the formation of proliferative diabetic membranes.

Ventricular expression and circulating levels of insulin-like growth factor I in heart transplant recipients.

1. Insulin-like growth factor I is a major mediator of growth-promoting activities. We studied the ventricular insulin-like growth factor I gene expression at mRNA and peptide levels in 24 heart transplant recipients (14 children and 10 adults), using 'slot blot' hybridization with insulin-like growth factor I cDNA probe and a specific radioimmunoassay. 2. Ventricular insulin-like growth factor I mRNA was detected in all the cardiac transplant children but was below the limit of detection in the cardiac transplant adults. Ventricular insulin-like growth factor I levels were significantly higher in the transplant children [174 +/- 15 (SEM; range 39-950) pg/mg soluble protein] than in transplant adults [39 +/- 2 (range 14-85) pg/mg soluble protein, P < 0.01, n = 14]. Circulating levels of insulin-like growth factor I in the cardiac transplant children [164 +/- 10 (range 105-192) ng/ml] and adults [176 +/- 15 (range 126-244) ng/ml] were within normal ranges for children and adults. 3. These results suggest that the human heart is a site for insulin-like growth factor I production and provide support for an autocrine role for insulin-like growth factor I in the ventricle, despite cardiac denervation.

Molecular forms of brain and atrial natriuretic peptides in transplanted human heart.

We examined the molecular forms of brain natriuretic peptide (BNP) and atrial natriuretic peptide (ANP) in endomyocardial right ventricular extracts from cardiac transplant recipients, using gel chromatographic separation coupled with specific radioimmunoassay techniques. Two distinct molecular forms of ANP and BNP were observed in the ventricular extracts consistent with the biologically active alpha-human peptides and the gamma-precursor peptides. Alpha-BNP was predominant and constituted 75 +/- 3% (n = 5) of the total immunoreactive BNP, while 21 +/- 2% (n = 5) of the total immunoreactivity comprised gamma-BNP. In contrast, gamma-ANP constituted 89 +/- 4% (n = 5) of the total immunoreactive ANP and was dominant over alpha-ANP, which made up 10 +/- 2% (n = 5) of the total immunoreactivity. There was no evidence for altered processing of BNP and ANP precursors in the right ventricular tissues from the de-nervated transplanted human heart. These findings provide further insight by identifying the molecular forms of BNP and ANP present in the transplanted human heart.

Natriuretic peptides and their receptors in human neural retina and retinal pigment epithelium.

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) play a role in transepithelial fluid movement in the kidney and at the blood-aqueous barrier. We sought a similar natriuretic peptide-mediated regulatory system at the level of the blood-retinal barrier by investigating human neural retina and retinal pigment epithelium for the presence of ANP, BNP and their receptors. ANP and BNP binding to receptors could be demonstrated autoradiographically in all layers of the retina inclusive of the retinal pigment epithelium. Competitive preincubation with unlabeled peptides blocked the binding of the respective radioactive peptide. ANP and BNP could also be demonstrated immunohistochemically in both the neural retina and the retinal pigment epithelium. Our results suggest a role of these peptides both in the regulation of intraretinal fluid movement and--by analogy with other peptides--as possible neutrotransmittors. The localisation of ANP and BNP in the retinal pigment epithelium suggests that these peptides may influence ocular fluid homeostasis at the outer blood-retinal barrier by modulating pigment epithelial function.

Secretion of atrial and brain natriuretic peptides from human cardiac atrial explants in culture: effect of dynorphin.

We have previously developed a method for maintaining human cardiac explants in culture under serum-free conditions, for the assessment of cardiac endocrine function and myocardial growth factors. In order to assess the local role of dynorphin in the human heart, we studied the effects of dynorphin on the secretion of atrial natriuretic peptide and brain natriuretic peptide by human cardiac atrial explants. Dynorphin did not affect the basal secretion of brain natriuretic peptide, but clearly enhanced the release of atrial natriuretic peptide from the human cardiac explants in culture. The atrial content of brain natriuretic peptide was not significantly reduced, whereas the atrial content of atrial natriuretic peptide in cultured explants was reduced two-fold in the presence of dynorphin. These findings indicate that dynorphin may have a direct stimulatory effect on the release of atrial natriuretic peptide, but not brain natriuretic peptide, from human cardiac atria.

Ventricular expression of basic fibroblast growth factor gene after orthotopic cardiac transplantation.

We studied the ventricular expression of basic fibroblast growth factor (bFGF) at the mRNA level in normal and transplanted human hearts as a direct measure of local bFGF gene expression. By Northern blot hybridization with a human bFGF cDNA probe, mRNA transcripts 7.5, 3.5, and 2.0 kb for bFGF were detected in both normal and transplanted hearts. Densitometric analysis of the transcripts after normalization for RNA loading revealed a 2-fold increase in the transplanted compared with the normal hearts. Slot blot hybridization of the ventricular RNA showed a significant increase in bFGF mRNA level in the transplanted over the normal hearts (P < 0.01) and was independent of hemodynamic parameters and immunosuppressive drugs. These results provide unequivocal evidence that the human heart is a site of bFGF production as demonstrated by the presence of bFGF mRNA. The increased ventricular expression of bFGF gene after heart transplantation may be of importance in the mediation of growth and repair of myocardial injury.

Ventricular expression of brain natriuretic peptide gene following orthotopic cardiac transplantation in children--a three year follow up.

OBJECTIVE: The aim was to examine ventricular brain natriuretic peptide (B-type natriuretic peptide, BNP) gene expression and to determine its relationship with ventricular BNP and circulating BNP levels in paediatric cardiac transplant recipients, over a three year period after transplantation. METHODS: Total RNA extracted from endomyocardial right ventricular biopsy tissues (n = 26) of 13 cardiac transplant recipients (age range 5-17 years) and 10 normal hearts obtained at necropsy (age range 19-76 years) as controls was analysed by northern and slot blot hybridisations. Specific radioimmunoassay techniques were used to determine levels of BNP and atrial natriuretic peptide (A-type natriuretic peptide, ANP) in plasma (n = 26) and ventricular biopsy (n = 26) samples. RESULTS: Ventricular BNP messenger ribonucleic acid (mRNA) levels from slot blot hybridisations in the transplanted heart [122(3) arbitrary units, range 97-143] were significantly higher (p < 0.01) than in the normal heart [63(5) arbitrary units, range 37-98]. Northern blot hybridisations confirmed this result and gave a major BNP mRNA transcript of approximately 900 nucleotides. There was no significant relationship between ventricular BNP mRNA levels and ventricular BNP (r = 0.15, p = 0.5, n = 26) or plasma BNP levels (r = 0.16, p = 0.4, n = 26). There was also no significant relationship between ventricular BNP mRNA levels and any of the haemodynamic variables, or immunosuppressive drugs. A ventricular ANP RNA transcript of approximately 900 nucleotides was detected in the transplanted heart but was below the limit of detection in the normal heart. For the long term study, increased levels of BNP and ANP in both plasma and ventricular samples were observed in the first year after transplantation, with a significant reduction (p < 0.01) in levels three years later. CONCLUSIONS: Ventricular BNP gene expression is increased at the mRNA level after heart transplantation in children. Expression of both ventricular BNP mRNA and ANP mRNA in the transplanted heart may be an important response in the modulation of cardiac function after transplantation.