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PubMed2003

Value of determining carbohydrate-deficient transferrin isoforms in the diagnosis of alcoholic liver disease.

OBJECTIVE: To determine whether isoform separation of carbohydrate-deficient transferrin (CDT) is of value in the diagnosis of alcoholic liver disease (ALD) and is specific to ALD when compared with other liver diseases. PATIENTS AND METHODS: During 1995 and 1996, 47 patients with ALD were evaluated with CDT at the Mayo Clinic in Rochester, Minn. The diagnosis of ALD was based on biochemical and histological analyses and on a history of drinking that exceeded 5 years with an average alcohol intake of more than 60 g/d. Disease controls included nonalcoholic steatohepatitis (NASH) (n = 26) and other liver disease (n = 22). Normal controls (n = 21) were healthy individuals without liver disease. Transferrin isoforms were quantified by densitometry of Coomassie-stained transferrins after affinity purification and isoelectric focusing. The pentasialo, tetrasialo, trisialo, disialo, monosialo, and asialo isoforms were quantified as percentages of total band densities. RESULTS: Receiver operating characteristic (ROC) curves were constructed for each isoform. The curves for total desialated isoforms (sum of disialo, monosialo, and asialo) displayed the best relationship between sensitivity and specificity with an ROC-area under the curve (AUC) of 0.922. The ROC-AUC values for individual transferrin isoforms in ALD vs NASH for pentasialo, tetrasialo, trisialo, disialo, monosialo, and asialo were 0.806, 0.917, 0.885, 0.933, 0.804, and 0.785, respectively. Only 58% of patients with ALD were detected at a specificity that excluded ALD in 84% of those who did not have it. CONCLUSION: Within alcohol ingestion times reported to us, no associations with recent drinking were observed. Alcohol as a cause of liver disease is not perfectly established by CDT analysis, although a high total CDT value favors ALD over NASH.

Adult↗
PubMed2004

RNA interference: a potential strategy for isoform-specific phosphatidylinositol 3-kinase targeted therapy in ovarian cancer.

Phosphatidylinositol 3-kinase (PI3-kinase) is a novel intracellular transducer involved in a wide range of cancer-associated signaling pathways, which comprises various isoforms and splice variants with distinct biologic activities and clinical implications. Especially, the class Ia PI3-kinase 110 kD catalytic subunit alpha (PIK3CA) is the most important isoform in tumorigenesis and possibly, tumor angiogenesis. Several strategies have been developed to block PI3-kinase for cancer therapy; however, the approach to target specific PI3-kinase isoform has not been explored to date. In the present study, we show that RNA interference (RNAi) through small interfering (siRNA) sequences targeting PIK3CA has potential applications in isoform-specific "knock-down" of PI3-kinase. This strategy provides a novel tool to study the function of various PI3-kinase isoforms and may contribute to isoform-specific targeting of PI3-kinase in human cancer.

Animals↗
PubMed1999

Expression and function of leptin receptor isoforms in myeloid leukemia and myelodysplastic syndromes: proliferative and anti-apoptotic activities.

The receptor for the gene product of the obesity gene, leptin, was recently reported to be expressed on murine and human hematopoietic progenitor cells. Therefore, we studied the expression of the leptin receptor, OB-R, in normal myeloid precursors, human leukemia cell lines, and primary leukemic cells using reverse-transcriptase polymerase chain reaction. In normal hematopoiesis, OB-R was expressed in CD34(+) cells. Normal promyelocytes (CD34(-)33(+) and CD34(-)13(+)) expressed only very low levels of the short, presumably nonsignaling isoform. Both the long and short isoforms of OB-R were expressed in 10 of 22 samples from patients with newly diagnosed primary or secondary acute myeloid leukemia (AML), with a higher incidence of the long isoform in primary AML (87.6% v 28.6%; P =.01). The incidence of OB-R expression was higher in recurrent than in newly diagnosed AML (P <.001), and samples from four patients with refractory AML showed strong expression of both isoforms. Both OB-R isoforms were also expressed in newly diagnosed and recurrent acute promyelocytic leukemia cells but were essentially absent in samples of chronic or acute lymphocytic leukemia. In vitro growth of myeloid leukemic cell lines and of blasts from 14 primary AMLs demonstrated that recombinant human leptin alone induced low level proliferation, significantly (P <.05) increased proliferation induced by recombinant human granulocyte colony-stimulating factor, interleukin 3, and stem cell factor in a subset of AML and increased colony formation (P <.005). Also, leptin reduced apoptosis induced by cytokine withdrawal in MO7E and TF-1 cells. Serum leptin levels correlated only with body mass index (P <. 001) and gender (P =.03). Results confirm the reported expression of leptin receptor in normal CD34(+) cells and demonstrate the frequent expression of leptin receptors in AML blasts. While normal promyelocytes lack receptor expression, leukemic promyelocytes express both isoforms. We also demonstrate proliferative effects of leptin alone and in combination with other physiologic cytokines, and anti-apoptotic properties of leptin. These findings could have implications for the pathophysiology of AML.

Apoptosis↗
PubMed1999

SNAP-25a and -25b isoforms are both expressed in insulin-secreting cells and can function in insulin secretion.

The tSNARE (the target-membrane soluble NSF-attachment protein receptor, where NSF is N-ethylmaleimide-sensitive fusion protein) synaptosomal-associated protein of 25 kDa (SNAP-25) is expressed in pancreatic B-cells and its cleavage by botulinum neurotoxin E (BoNT/E) abolishes stimulated secretion of insulin. In the nervous system, two SNAP-25 isoforms (a and b) have been described that are produced by alternative splicing. Here it is shown, using reverse transcriptase PCR, that messages for both SNAP-25 isoforms are expressed in primary pancreatic B and non-B cells as well as in insulin-secreting cell lines. After transfection, both isoforms can be detected at the plasma membrane as well as in an intracellular perinuclear region in the insulin-secreting cell line, HIT. To test for the functional role of the two isoforms in insulin secretion, mutant forms of SNAP-25a and b resistant against cleavage by BoNT/E were generated. Such mutant SNAP-25, when expressed in HIT cells, is not inactivated by BoNT/E and its ability to restore insulin secretion can thus be investigated. To obtain the toxin-resistant mutant isoforms, the sequence around the BoNT/E cleavage site (R176QIDRIM182) was changed to P176QIKRIT182. This is the sequence of the equivalent region of human SNAP-23 (P187-T194), which has been shown to be resistant to BoNT/E. The mutant SNAP-25 was resistant to BoNT/E in vitro and in vivo and both mutant isoforms were able to reconstitute insulin secretion from toxin-treated HIT cells.

Animals↗
PubMed1999

Expression of genes for microphthalmia isoforms, Pax3 and MSG1, in human melanomas.

Microphthalmia (MITF) gene product, a transcription factor of the basic-helix-loop-helix type, is thought to play a role in the regulation of genes encoding the enzymes necessary for melanogenesis. These include tyrosinase, TRP-1 and TRP-2. Melanocyte-specific isoform of microphthalmia, MITF-M, is expressed in normal and malignant melanocytes. The presence of two other isoforms of microphthalmia, MITF-A and MITF-H, which differ from MITF-M in the amino-terminus, was demonstrated also in some non-melanocytic lineages. Here we have analyzed the presence of all three known isoforms of MITF mRNA in a panel of 17 human melanoma cell lines by a reverse transcriptase-polymerase chain reaction using isoform-specific primers. While, as expected, the predominant form in melanoma cell lines was MITF-M, low amounts of MITF-A mRNA was found in almost all melanomas, as well as in most of 20 tumor cell lines of the non-melanocyte origin (lung and colon carcinomas, osteosarcomas and neuroblastomas). The expression of MITF-H was not detected, with a few exceptions, in the tested cell lines. Pax3 transcription factor was reported earlier to regulate positively the melanocyte-specific promoter of the MITF gene. We found here that the Pax 3 mRNA was expressed in all melanoma cell lines, even in those that had repressed the MITF-M and were amelanotic. This suggests that additional factors, besides Pax3, are required for the MITF expression. The MSG1 (melanocyte-specific gene 1), a gene originally isolated from melanocytes and containing a strong transcription activation domain, was also found expressed in all melanomas and most non-melanocyte tumor cell lines. Together, these data indicate that the MITF-M isoform is the major type of MITF mRNA present in human melanoma cell lines and show that the expression of the isoform MITF-A and the MSG1 is not restricted to malignant melanocytes and occurs in a wide range of tumor cell lines.

DNA-Binding Proteins↗
PubMed2000

Tropomyosin isoform 5b is expressed in human erythrocytes: implications of tropomodulin-TM5 or tropomodulin-TM5b complexes in the protofilament and hexagonal organization of membrane skeletons.

The human erythrocyte membrane skeleton consists of hexagonal lattices with junctional complexes containing F-actin protofilaments of approximately 33-37 nm in length. We hypothesize that complexes formed by tropomodulin, a globular capping protein at the pointed end of actin filaments, and tropomyosin (TM), a rod-like molecule of approximately 33-35 nm, may contribute to the formation of protofilaments. We have previously cloned the human tropomodulin complementary DNA and identified human TM isoform 5 (hTM5), a product of the gamma-TM gene, as one of the major TM isoforms in erythrocytes. We now identify TM5b, a product of the alpha-TM gene, to be the second major TM isoform. TM5a, the alternatively spliced isoform of the alpha-TM gene, which differs by 1 exon and has a weaker actin-binding affinity, however, is not present. TM4, encoded by the delta-TM gene, is not present either. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, hTM5 comigrated with the slower TM major species in erythrocyte membranes, and hTM5b comigrated with the faster TM major species. TM5b, like TM5, binds strongly to tropomodulin, more so than other TM isoforms. The 2 major TM isoforms, therefore, share several common features: They have 248 residues, are approximately 33-35 nm long, and have high affinities toward F-actin and tropomodulin. These common features may be the key to the mechanism by which protofilaments are formed. Tropomodulin-TM5 or tropomodulin-TM5b complexes may stabilize F-actin in segments of approximately 33-37 nm during erythroid terminal differentiation and may, therefore, function as a molecular ruler. TM5 and TM5b further define the hexagonal geometry of the skeletal network and allow actin-regulatory functions of TMs to be modulated by tropomodulin. (Blood. 2000;95:1473-1480)

Amino Acid Sequence↗
PubMed2000

Improved contractility of obstructed bladders after Tadenan treatment is associated with reversal of altered myosin isoform expression.

PURPOSE: Tadenan is a plant extract from Pygeum africanum used in the treatment of benign prostatic hyperplasia, to protect the bladder from contractile dysfunction induced by partial bladder outlet obstruction (BOO). The aim of the present study was to determine whether the Tadenan-induced return of detrusor contractility affects the expression of myosin isoforms, which differ at the C-terminal (SM1 and SM2) and the N-terminal regions (SM-A and SM-B). MATERIALS AND METHODS: Four groups of New Zealand White rabbits (3 to 5 kg., 4 to 6 rabbits per group) were either partially obstructed by ligation of the urethra (groups 1 and 2) or not obstructed (groups 3 and 4). After 2 weeks, rabbits from groups 2 and 4 received Tadenan in peanut oil (vehicle) orally at 100 mg. /kg./day for 3 weeks and rabbits in groups 1 and 3 received vehicle only. Rabbits were sacrificed and bladders were removed and weighed. Contractility studies were performed on isolated strips of detrusor and the remaining muscular layer from the bladder body was used to study the expression of myosin heavy chain (MHC) isoforms at mRNA (SM1, SM2, SM-A, and SM-B) and the protein (SM1 and SM2) levels by RT-PCR and SDS-PAGE analyses, respectively. RESULTS: Tadenan significantly reduced the effect of BOO on bladder mass. The diminished contractile response to field stimulation and carbachol secondary to urethral obstruction was significantly reversed by Tadenan treatment. The relative ratios for MHC isoforms were altered at the mRNA (SM2:SM1 and SM-A:SM-B) and protein (SM2:SM1) levels in obstruction. Upon treatment with Tadenan, the ratio of these isoforms returned to normal, as shown at the mRNA levels. In addition, the altered relative ratio of SM2:SM1 at the protein level also returned to nearly normal values after treatment. CONCLUSIONS: Improvement of obstruction-induced contractile dysfunction of the detrusor following treatment with Tadenan is associated with changes in the expression of myosin isoforms. The alteration in the expression of myosin isoforms associated with obstruction-induced hypertrophy is reversed close to normal in the detrusor smooth muscle from Tadenan-treated obstructed rabbits.

Animals↗
PubMed2000

Lipidation of apolipoprotein E influences its isoform-specific interaction with Alzheimer's amyloid beta peptides.

The inheritance of the apolipoprotein E (apoE) epsilon4 allele is a prevailing risk factor for sporadic and familial Alzheimer's disease (AD). ApoE isoforms bind directly to Alzheimer's amyloid beta (Abeta) peptides both in vitro and in vivo. Recent studies suggest that association of apoE with lipids may modulate its interaction with Abeta. We examined the binding of lipid-associated and delipidated apoE3 and apoE4 isoforms to Abeta utilizing a solid-phase binding assay and estimated the dissociation constants for the interaction of various apoE and Abeta species. Using native apoE isoforms from stably transfected RAW 264 and human embryonic kidney 293 cells, apoE3 had greater affinity than apoE4 for both Abeta1-40 and Abeta1-42. Delipidation of apoE decreased its affinity for Abeta peptides by 5-10-fold and abolished the isoform-specificity. Conversely, incorporation of apoE isoforms produced by baculovirus-infected Sf9 cells into reconstituted human high-density-lipoprotein lipoparticles restored the affinity values for Abeta peptides and resulted in preferential binding of apoE3. The data demonstrate that native lipid-associated apoE3 binds to Abeta peptides with 2-3-fold higher affinity than lipid-associated apoE4. Since the isoforms' binding efficiency correlate inversely with the risk of developing late-onset AD, the results suggest a possible involvement of apoE3 in the clearance or routing out of Abeta from the central nervous system as one of the mechanisms underlying the pathology of the disease.

Amyloid beta-Peptides↗
PubMed2000

Translational control of C/EBPalpha and C/EBPbeta isoform expression.

Transcription factors derived from CCAAT/enhancer binding protein (C/EBP)alpha and C/EBPbeta genes control differentiation and proliferation in a number of cell types. Various C/EBP isoforms arise from unique C/EBPbeta and C/EBPalpha mRNAs by differential initiation of translation. These isoforms retain different parts of the amino terminus and therefore display different functions in gene regulation and proliferation control. We show that PKR and mTOR signaling pathways control the ratio of C/EBP isoform expression through the eukaryotic translation initiation factors eIF-2alpha and eIF-4E, respectively. An evolutionary conserved upstream open reading frame in C/EBPalpha and C/EBPbeta mRNAs is a prerequisite for regulated initiation from the different translation initiation sites and integrates translation factor activity. Deregulated translational control leading to aberrant C/EBPalpha and C/EBPbeta isoform expression or ectopic expression of truncated isoforms disrupts terminal differentiation and induces a transformed phenotype in 3T3-L1 cells. Our results demonstrate that the translational controlled ratio of C/EBPalpha and C/EBPbeta isoform expression determines cell fate.

Adipocytes↗
PubMed2000

CD97 isoform expression in leukocytes.

Different adhesive capacity in interactions with CD55 has been ascribed to the isoforms of the leukocyte CD97 antigen, CD97 (EGF 1,2,5), CD97 (EGF 1,2,3,5), and CD97 (EGF 1,2,3,4,5). In the study, coexpression of the three CD97 isoforms and predominance of CD97 (EGF 1,2,5) transcripts in leukocytes are demonstrated. The contribution of CD97 (EGF 1,2,3,5) and CD97 (EGF 1,2,3,4,5) to total CD97 levels varied among most cell types only slightly, although relatively higher mRNA levels of both isoforms were detected in U 937 cells and monocytes. In peripheral blood lymphocytes, CD97 isoforms did not show clear variation after PMA stimulation and were down-regulated equally after CD97 cross-linking. Moreover, the CD97 isoform pattern was not altered in monocytes after interferon-gamma stimulation and in synovial T cells from patients with rheumatoid arthritis. CD97 mRNA levels did not necessarily correspond to CD97 surface density. The findings suggest that adhesive activity of CD97 toward CD55 is unlikely to be regulated by differential CD97 isoform expression.

Antigens, CD↗
PubMed2000

Characterization and functional analysis of laminin isoforms in human bone marrow.

Laminins are a family of disulfide-linked heterotrimeric proteins consisting of 3 different subunits termed alpha, beta, and gamma chains. Combinations of 11 characterized laminin subunits (alpha 1-alpha 5, beta 1-beta 3, and gamma 1-gamma 3) generate at least 12 laminin isoforms, which can serve different functions. Although expression of laminin in the hematopoietic microenvironment has been known for many years, the nature of the laminin isoforms present in the human bone marrow is poorly characterized. The present study attempts to clarify this issue. Reverse transcriptase-polymerase chain reaction analysis of human bone marrow stromal cells suggested the expression of many laminin isoforms in the marrow. Northern blot and immunoblot analysis, however, showed that laminin-8/9 and laminin-10/11 are the most abundant laminin isoforms synthesized by human bone marrow stromal cells. Other isoforms, if present, certainly play a minor role in the hematopoietic microenvironment. Functionally, laminin-10/11 preparations showed strong adhesive interactions with human CD34(+) cell lines. Antibodies against the beta 1 integrin subunit inhibited these interactions. Other laminin isoforms, especially laminin-1 and laminin-2/4, showed only weak or no adhesive interactions with the hematopoietic cell lines tested, explaining former negative results. In addition to its adhesion-mediating properties, laminin-10/11 preparations also showed a mitogenic activity for human hematopoietic progenitor cells. Taken together, these data suggest that laminin in the bone marrow plays a hitherto unexpected important function in the development of hematopoietic progenitor cells. (Blood. 2000;96:4194-4203)

Blotting, Western↗
PubMed2000

Serum leptin and leptin receptor isoforms in omental adipose tissue of nondiabetic women undergoing gynecologic surgery for benign disease.

BACKGROUND AND PURPOSE: Leptin is important in the regulation of fat mass and body weight. Adipose tissue not only secretes leptin but also serves as a site of action for leptin. This study was designed to examine the relationships among tissue expression of leptin receptors, serum leptin, and body mass index. METHODS: Omental adipose tissue and fasting blood samples were obtained from 57 nondiabetic women who underwent surgery for either myoma of the uterus or ovarian cyst. Tissue RNA was extracted using Trizol reagent and serum leptin concentrations were determined with commercial kits. The leptin receptor isoforms in tissues were quantified using real-time Taqman technology. RESULTS: Three leptin receptor isoforms, Ob-Rb, HuB219.1, and HuB219.3, were found in human omental adipose tissue. The amounts of HuB219.1 and HuB219.3 mRNA relative to that of Ob-Rb were 1314.2 and 16.7, respectively. Higher body mass index was significantly correlated with an increase in serum leptin concentration and a decrease in leptin receptor HuB219.1 isoform in omental fat, even after adjustment for age and menopausal status. There was no direct association between serum leptin concentration and tissue HuB219.1 mRNA level. CONCLUSIONS: HuB219.1 is the major isoform of leptin receptor expressed in human omental adipose tissue. Our findings suggest that the shorter leptin receptor isoforms in human omental adipose tissue might play an important role in body weight control. Further studies on the inter-relationship between leptin concentrations and multiple leptin receptor isoforms are needed to elucidate the exact mechanism of obesity.

Adipose Tissue↗
PubMed2001

Rapid determination of transferrin isoforms by immunoaffinity liquid chromatography and electrospray mass spectrometry.

BACKGROUND: Congenital disorders of glycosylation (CDG) are autosomal recessive disorders that produce increased serum carbohydrate-deficient transferrin (CDT) isoforms. Methods to resolve CDT from fully glycosylated transferrin (Trf) have been based on a neutral shift in the isoelectric focusing (IEF) pattern or on a reduction in the negative charge, allowing resolution by anion-exchange chromatography. Our purpose was to develop a method of resolution and relative quantification of Trf isoforms using online immunoaffinity liquid chromatography-mass spectrometry (LC-MS). METHODS: Serum (25 microL) was diluted with 100 microL of water before application to an immunoaffinity column that sequestered Trf isoforms. Trf isoforms were eluted from the immunoaffinity column, concentrated on a C4 column, eluted from the C4 column, and introduced into the mass spectrometer. Analysis of the Trf isoforms was entirely automated and completed in <10 min per sample. RESULTS: The LC-MS method demonstrated that the major abnormal Trf isoforms in CDG lack one complete oligosaccharide structure (mono-oligosaccharide) or both oligosaccharide structures (a-oligosaccharide), but not the sialic acids, as presumed on the basis of IEF methods. Calculation of relative ratios among three possible species (mono-/di-oligosaccharide and a-/di-oligosaccharide) is reproducible [mean intra- and interassay CVs were 9.3% (n = 10) and 10% (n = 5), respectively]. A reference range for patients <18 years was determined by analysis of 209 samples (for mono-/di-oligosaccharide, the median was 0.041 and the range was 0.018-0.083; for a-/di-oligosaccharide, the median was 0.007 and the range was 0.002-0.036). Comparison of data obtained with an affinity chromatography-IEF method and the LC-MS method demonstrated equivalence in the interpreted results (n = 170). CONCLUSIONS: Advantages of the LC-MS method include improved sensitivity, minimal sample preparation, and an analysis time of <10 min. The method was automated, which allowed high throughput, with >100 samples analyzed in a single day. Moreover, the nature of the oligosaccharide defect in CDG is accurately reflected by mass resolution, and subtle oligosaccharide truncations may also be detected by this method.

Animals↗
PubMed2001

Differential control of gene activity by isoforms A, B1 and B2 of the Drosophila ecdysone receptor.

The steroid hormone ecdysone initiates molting and metamorphosis in Drosophila via a heterodimeric receptor consisting of EcR that binds hormone, and USP, a homolog of the vertebrate RXR receptor. EcR exists in three isoforms EcRA, EcRB1 and EcRB2 that are thought to direct specific physiological responses to ecdysone. These three isoforms differ only in their N-terminal A/B domain that implies that sequences responsible for the differential physiological effects lie within the A/B domains of the EcR isoforms. In the present study, we set out to determine the capability of the three isoforms and their A/B domains to control gene transcription. When full-length EcR plasmids were cotransfected into mammalian cells with a USP expressing and a cognate reporter plasmid, the three EcR isoforms showed striking differences in their ability to control gene transcription, both in the presence and in the absence of hormone. Furthermore, the A/B domains of EcRB1 and of EcRB2 when fused to the GAL4 DNA binding domain are sufficient to activate transcription of a reporter gene, in yeast as well as in mammalian cells. In contrast, a fusion construct containing the A/B domain of EcRA represses basal transcription of the reporter gene. All these findings emphasize the importance of the A/B domains of the three EcR isoforms for differentially controlling gene transcription. Furthermore, they provide evidence for the existence of an autonomous ligand-independent activation function (AF1) in the A/B domains of EcRB1 and EcRB2 and of an inhibitory function (IF) in the A/B domain of EcRA.

Amino Acid Sequence↗
PubMed2001

Impact of apolipoprotein(a) isoform size heterogeneity on the lysine binding function of lipoprotein(a) in early onset coronary artery disease.

Elevated plasma Lp(a) is an independent risk factor for cardiovascular disease. Unique to Lp(a) is the apoprotein, apo(a) which can vary from 250 to 800 kDa in molecular weight. Small isoforms are also associated with the risk of cardiovascular disease. The purpose of this study was to examine the association of Lp(a) concentration, apo(a) size, and Lp(a) lysine-binding site(s) (LBS) function in patients with early onset heart disease, and age-matched controls. Mean values of Lp(a) were significantly higher in the patients than for the age-matched group. The smallest molecular weight isoform for each subject had significantly fewer kringles for the patients than the age-matched controls. There was a significant correlation between LBS activity and kringle number in the single-banded phenotypes of the patients, but not the controls. LBS activity was significantly higher in patients with small isoforms (< or =18 kringles) compared to controls. The odds ratio for coronary artery disease for high LBS activity and high Lp(a) concentration was 4.4 (p = 0.002) and for high LBS activity and small isoforms was 10.1 (p = 0.002). In the patients, Lp(a) concentration was higher, apo(a) size was smaller, and LBS activity higher in the small isoforms compared to the controls. This study suggests an association of high LBS activity in small isoforms of Lp(a) with disease in humans.

Adult↗
PubMed2001

Vascular endothelial growth factor isoforms display distinct activities in promoting tumor angiogenesis at different anatomic sites.

The gene for the major angiogenic factor, vascular endothelial growth factor (VEGF), encodes several spliced isoforms. We reported previously that overexpression of two VEGF isoforms, VEGF(121) and VEGF(165), by human glioma U87 MG cells induced tumor-associated intracerebral hemorrhage, whereas expression of a third form, VEGF(189), did not cause vessel rupture. Here, we test whether these VEGF isoforms have distinct activities for enhancing vascularization and growth of gliomas in mice. U87 MG cells that overexpressed VEGF(165) or VEGF(189) grew more rapidly than the parental cells in both s.c. and intracranial (i.c.) locations. However, cells that overexpressed VEGF(121) only showed enhancement of i.c. tumor growth but had a minimal effect on s.c. glioma progression. At both anatomical sties, VEGF(165) and VEGF(189) strongly augmented neovascularization, whereas VEGF(121) only increased vessel density in brain tumors. In each type of glioma, expression of VEGF receptors -1 and -2 largely phenocopied the tumor vasculature, because increased VEGF/VEGF receptor-activated microvessel densities were strongly correlated with the angiogenicity and tumorigenicity elicited by the VEGF isoforms at both anatomical sites. One notable difference between the sites was the expression of vitronectin, a prototypic ligand of alpha(v)beta(3) and alpha(v)beta(5) integrins, detected in i.c. but not in s.c., gliomas. Endothelial cell migration stimulated by VEGF(121) was potentiated by vitronectin to a greater extent than that stimulated by VEGF(165). This data demonstrates that VEGF isoforms have distinct activities at different anatomical sites and suggest that the microenvironment of different tissues affects the function of VEGF isoforms.

Animals↗
PubMed2002

Apolipoprotein [a] genotype influences isoform dominance pattern differently in African Americans and Caucasians.

Plasma lipoprotein [a] (Lp[a]) concentrations are inversely associated with, and largely determined by, apolipoprotein [a] (apo[a]) gene size, a highly polymorphic trait. We studied if, within an individual, the smaller apo[a] isoform always dominated, whether there was interaction between the two alleles, and whether these features differed between Caucasians and African Americans. We determined apo[a] gene sizes, apo[a] protein sizes and relative amounts, and plasma Lp[a] levels in 430 individuals (263 Caucasians and 167 African Americans). Of the 397 heterozygotes with at least one detectable apo[a] isoform (238 Caucasians and 159 African Americans), the larger allele dominated in 28% of Caucasians and 23% of African Americans, while the smaller allele dominated in 56% of Caucasians and 45% of African Americans. In Caucasians, dominance of the smaller allele increased with Lp[a] levels, from 44% at Lp[a] < or = 30 nM to 81% at Lp[a] >100 nM (P < 0.0001). Dominance by the smaller allele increased with increasing size of the larger allele in both groups but with the smaller allele only in African Americans. There was no interaction between apo[a] alleles within genotypes; one apo[a] isoform level was not associated with the other isoform level, and isoform levels were not affected by the difference in size. More of the dominance pattern was explained by Lp[a] level and apo[a] genotype in African Americans than in Caucasians (29% vs. 13%). Thus, genotype influences isoform-specific Lp[a] levels and dominance patterns differently in African Americans and in Caucasians.

Aged↗
PubMed2002

Vascular endothelial growth factor isoform expression as a determinant of blood vessel patterning in human melanoma xenografts.

Vascular endothelial growth factor (VEGF) occurs in at least five different isoforms because of alternative splicing of the gene. To investigate the roles of different VEGF isoforms in tumor blood vessel formation and tumorigenicity, the three major isoforms (VEGF(121), VEGF(165), and VEGF(189)) were overexpressed in an early-stage human melanoma cell line (WM1341B), which is VEGF-negative and nontumorigenic in immunodeficient mice. Although overexpression of VEGF(121) and VEGF(165) resulted in aggressive tumor growth, WM1341B cells transfected with VEGF(189) remained nontumorigenic and dormant on injection. Although tumor growth rate depended on the level and not the isoform of VEGF expressed, striking isoform-specific differences in vascular patterning were associated with VEGF(121)- versus VEGF(165)-dependent tumorigenic conversion of human melanoma. Thus, tumors overexpressing VEGF(165) generated dense, highly heterogeneous vessel networks that were distinctly different from those of tumors expressing VEGF(121) (poorly vascularized and necrotic). Paradoxically, although VEGF(165) expression appears to result in the most effective tumor perfusion, it is the expression of VEGF(121) that is observed during human malignant melanoma progression. Indeed, unbiased selection of spontaneously tumorigenic variants of WM1341B (by coinjection with Matrigel) led to predominant expression of the VEGF(121) isoform. The vascular patterning in these tumors (1341-P3N1, 1341-P3N2) resembled that of the VEGF(121)-transfected WM1341B tumors. These results suggest that, for reasons that remain to be elucidated, a "minimal" program of tumor vascularization may be favored during melanoma progression.

Animals↗