PubMed Health⌕ Search

Biomedical subjects

M B Grant

Publications and source records attributed to M B Grant.

At least 37 records · Page 2Linked to original sources
PubMed1996

Expression of IGF-I, IGF-I receptor and IGF binding proteins-1, -2, -3, -4 and -5 in human atherectomy specimens.

The molecular and cellular processes that induce rapid atherosclerotic plaque progression in patients with unstable angina and initiate restenosis following coronary interventional procedures are uncertain. We examined primary (de novo) and restenotic lesions retrieved at the time of directional coronary atherectomy for expression of insulin-like-growth factor-I (IGF-I). IGF-I receptor, and five IGF binding proteins (IGFBPs), IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-5 in smooth muscle cells (SMCs) using colloidal gold immunocytochemistry. IGF-1, its receptor and binding proteins were not detected in SMCs of normal coronary arteries. IGF-I localized primarily in synthetic smooth muscle cells (sSMCs) in both de novo and restenotic plaques. IGF-I receptor localized on sSMCs and their processes and colocalized with IGF-I. Although morphometric analysis of IGF-I and IGF-I receptor immunoreactivity in sSMCs of de novo and restenotic lesions showed comparable levels of IGF-I (3.2 +/- 1.0 and 2.9 +/- 0.9, respectively). IGF-I receptor was significantly higher in de novo lesions as compared to restenotic lesions (10.7 +/- 2.5 and 4.2 +/- 1.3, P < 0.05, respectively). IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4 and IGFBP-5 localized in the cytoplasm of sSMCs and in the extracellular matrix. Quantitative reverse transcription polymerase chain reaction (QRT-PCR) performed on de novo atherectomy specimens identified mRNA for IGF-I, IGF-I receptor, IGFBP-1, IGFBP-2, IGFBP-4, IGFBP-5 levels and detected mRNA for IGFBP-3. The expression of IGF-I, IGF-I receptor, and IGFBPs in atherectomy plaques suggests that the development of coronary obstructive lesions may be a result of changes in the IGF system.

Atherectomy, Coronary↗
PubMed1996

Plasminogen activator inhibitor-1 overexpression in nonproliferative diabetic retinopathy.

Plasminogen activator inhibitor-1 is secreted bidirectionally by endothelial cells, acts as the primary regulator of fibrinolysis and as a key modulator of extracellular matrix proteolysis. Elevated serum levels of plasminogen activator inhibitor-1 are observed in serum of diabetic individuals. We investigated whether plasminogen activator inhibitor-1 is overexpressed in capillaries of diabetic donors with non-proliferative retinopathy compared to non-diabetic donors. We also assessed plasminogen activator inhibitor-1 expression in an animal model of retinopathy induced by exposing rabbit retinas to insulin-like growth factor-I. Colloidal gold immunocytochemistry was used to quantify plasminogen activator-1 antigen in donor retinas from diabetic subjects (n = 10) and control subjects (n = 10). This technique was also used to examine expression of plasminogen activator inhibitor-1 for correlation with retinal changes in the insulin-like growth factor-I-induced retinopathy model (n = 14). Plasminogen activator inhibitor-1 immunoreactivity was significantly increased in the retinas of all diabetic subjects as compared to controls. In the rabbit model, the expression of plasminogen activator inhibitor-1 immunoreactivity correlated with pathological retinal changes. In both the diabetic human and insulin-like growth factor-I-injected rabbit, overproduction of plasminogen activator inhibitor-1 was seen within the lumen of capillaries, within the cytoplasm of endothelial cells and in the basement membrane and extracellular matrix surrounding these capillaries. Minimal plasminogen activator inhibitor-1 was detected in the retinas of non-diabetics and in control rabbits injected with either heat-inactivated insulin-like growth factor-I or balanced salt solution. These studies support the conclusion that plasminogen activator inhibitor-1 is overexpressed in the retinal capillaries of diabetics with non-proliferative diabetic retinopathy and in rabbits with insulin-like growth factor-I-induced retinopathy.

Adult↗
PubMed1996

Competitive RNA templates for detection and quantitation of growth factors, cytokines, extracellular matrix components and matrix metalloproteinases by RT-PCR.

Detection of low-abundance mRNAs by reverse transcription-polymerase chain reaction (RT-PCR) has become a standard technique to determine gene expression by tissues and cells in culture. The ability to determine relative or absolute copy number of specific mRNAs has been difficult due to inadequate internal standards to control for sample-to-sample variation. The use of a synthetic RNA standard with identical sequences to the PCR primers allows reproducible quantitation between samples and assays. By designing multi-sequence templates, several specific mRNAs can be quantitated using a single template. Addition of multiple templates to a single RT reaction allows the quantitation of a large number of targets from as little as 4 micrograms of total RNA. In this report, we present a series of seven primer/template systems to detect and quantitate 52 specific messages, including 26 growth factors and receptors, 8 extracellular matrix components, 10 matrix-modifying enzymes and their inhibitors and 8 cytokines.

Binding, Competitive↗
PubMed1994

Localization of insulin-like growth factor I and inhibition of coronary smooth muscle cell growth by somatostatin analogues in human coronary smooth muscle cells. A potential treatment for restenosis?

In this study, we demonstrate, for the first time, the localization of insulin-like growth factor I (IGF-I) in de novo and restenotic human coronary atherectomy plaques by using immunocytochemical techniques. Smooth muscle cells (SMCs) exhibiting the synthetic phenotype contained a statistically significant higher concentration of IGF-I than SMCs of the contractile phenotype or SMCs from normal coronary arteries. In addition, we provide data to suggest that the long-acting somatostatin analogues octreotide and angiopeptin inhibit IGF-I- and basic fibroblast growth factor (b-FGF)- induced human coronary artery SMC proliferation. Platelet-derived growth factor (PDGF)-stimulated cultures were minimally affected by the addition of octreotide but were significantly inhibited by angiopeptin. All three growth factors stimulated SMC migration in a dose-dependent manner. The somatostatin analogues tested had no effect on growth factor-stimulated SMC migration. Our data suggest that by reducing SMC proliferation, somatostatin analogues may have clinical usefulness in reducing the high incidence of restenosis observed after percutaneous transluminal coronary artery interventions.

Adult↗
PubMed1994

Differences in the synthesis of secreted proteins in human retinal endothelial cells of diabetic and nondiabetic origin.

Protein synthesis and deposition by vascular endothelial cells play an important role in the neovascularization seen in diabetic retinopathy. In the present study, we have compared the pattern of protein accumulation in human retinal endothelial cells derived from diabetic and nondiabetic individuals. Confluent cultures of retinal endothelial cells were incubated for 18 h with a mixture of radiolabeled methionine and cysteine. Under basal conditions, without the addition of growth factors, diabetic retinal endothelial cells accumulated less radiolabeled protein than did cells of nondiabetic origin. Both epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) enhanced protein accumulation in cells of diabetic origin, but not in cells of nondiabetic origin. Analysis of radiolabeled proteins in the secreted fraction by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) revealed prominent protein bands at 220 and 49.5 kD in both diabetic and nondiabetic cultures that were identified by immunoblot analysis as fibronectin and a mixture of secreted protein acidic and rich in cysteine (SPARC) and plasminogen activator inhibitor-1 (PAI-1), respectively. The levels of PAI-1 were higher in the secreted fractions of diabetic cultures than in nondiabetic cultures. SDS-PAGE and autoradiography of the secreted fraction also revealed two protein components of approximate molecular weight 440 and 78 kD, which were present in fractions of diabetic origin but absent in those of nondiabetic origin. Our studies support unique differences in protein expression in cells of diabetic vs. nondiabetic origin in response to EGF and bFGF and identify two proteins exclusively expressed by cells of diabetic origin.

Adult↗
PubMed1993

Insulin-like growth factor I acts as an angiogenic agent in rabbit cornea and retina: comparative studies with basic fibroblast growth factor.

The release of growth factors from ischaemic retina has been hypothesized as the central stimulus for retinal neovascularization in proliferative diabetic retinopathy. Two of the growth factors implicated are insulin-like growth factor-I and basic fibroblast growth factor. We examined the effect of insulin-like growth factor-I on in vivo neovascularization using the established angiogenic model of the rabbit cornea (n = 30), and also compared the effects of insulin-like growth factor-I and basic fibroblast growth factor using two new in vivo systems. Either supraphysiologic concentrations of each growth factor (600 micrograms) were injected intravitreally into pigmented rabbits (n = 21) or porous polyfluorotetraethylene chambers filled with an emulsion containing collagen and growth factor (500 ng) were placed on the retina surface (n = 8). Our results demonstrate that when insulin-like growth factor-I was implanted together with a slow release carrier into the pocket of the normally avascular cornea, insulin-like growth factor-I (10 micrograms/pellet) induced angiogenesis in all rabbits. This degree of angiogenesis was comparable to that previously shown for basic fibroblast growth factor. For the intravitreal studies, the fibrotic component was greater in the basic fibroblast growth factor injected eyes, whereas the vascular component was accentuated in the eyes injected with insulin-like growth factor-I. Light and electron microscopy demonstrated areas of vascular proliferation in both groups. Porous polyfluorotetraethylene chamber studies with insulin-like growth factor-I and basic fibroblast growth factor demonstrated vascular proliferation in the vicinity of the chamber similar to the intravitreal injected eyes, but to a lesser degree than the injected eyes. Our experiments overall support the angiogenic potential of both insulin-like growth factor-I and basic fibroblast growth factor and support distinct but complimentary roles for each growth factor in the pathogenesis of proliferative diabetic retinopathy.

Animals↗
PubMed1992

Treatment of alkali-injured rabbit corneas with a synthetic inhibitor of matrix metalloproteinases.

Healing of corneal alkali injuries remains a severe clinical challenge. The authors evaluated the effect of a new synthetic inhibitor of matrix metalloproteinases (GM6001 or N-[2(R)-2-(hydroxamido carbonylmethyl)-4-methylpentanoyl]-L-tryptophane methylamide) on preventing ulceration of rabbit corneas after alkali injury. Topical treatment of corneas with severe alkali injuries with 400 micrograms/ml or 40 micrograms/ml GM6001 alone prevented ulceration for 28 days, although 8 of 10 corneas treated with vehicle perforated. Corneas treated with 4 micrograms/ml GM6001 had midstromal depth ulcers. Corneas treated with 400 micrograms/ml of GM6001 contained very few inflammatory cells and had significantly reduced vessel ingrowth compared with vehicle-treated corneas. Epithelial regeneration after moderate alkali injuries also was investigated. Persistent epithelial defects developed 4 days after moderate alkali injury in rabbit corneas treated with vehicle and progressively increased to an average of 20% of the original 6 mm diameter wound by 27 days after moderate alkali injury. By contrast, epithelial regeneration was complete and persisted for 21 days for corneas treated with a formulation containing GM6001 (400 micrograms/ml), epidermal growth factor (10 micrograms/ml), fibronectin (500 micrograms/ml), and aprotinin (400 micrograms/ml). Sporadic punctate staining developed in 20% of the corneas treated with the combination of agents between days 21-28 after moderate alkali injury. These results demonstrate that topical application of GM6001 prevented corneal ulceration after severe alkali injury and that a combination containing GM6001, epidermal growth factor, fibronectin, and aprotinin promoted stable regeneration of corneal epithelium after moderate alkali injury.

Alkalies↗
PubMed1992

Effects of epidermal growth factor, fibroblast growth factor, and transforming growth factor-beta on corneal cell chemotaxis.

The effects of recombinant basic fibroblast growth factor (FGF), epidermal growth factor (EGF), and transforming growth factor-beta (TGF-beta) on migration of human and bovine corneal cells were determined using checkerboard analysis in Boyden chambers. EGF, FGF, and TGF-beta each stimulated high levels of chemotactic migration. Each growth factor, however, induced a different dose-response pattern. Migration stimulated by FGF reached a plateau at a concentration between 100 and 200 ng/ml for endothelial, epithelial, and stromal fibroblasts. By contrast, chemotactic responses to EGF peaked between 10 and 50 ng/ml, then decreased at higher concentrations. TGF-beta also stimulated a peak in migration in all three corneal cells, but the peak of migration occurred at an approximately 1000-fold lower concentration (1 pg/ml) than for EGF. Checkerboard analysis demonstrated that FGF and EGF, but not TGF-beta, stimulated chemokinesis of bovine, stromal, and endothelial cells. These results demonstrate that FGF, EGF, and TGF-beta induce migration in pure populations of bovine and human corneal cells and support the concept that these growth factors may play key roles in corneal wound healing by regulating migration of corneal cells.

Animals↗
PubMed1991

Neovascular growth factors.

Neovascularisation is the biological process of forming new blood vessels. Many conditions can initiate neovascularisation including trauma or chronic ischaemia produced by diseases such as diabetes. Neovascularisation proceeds through a series of steps beginning with destruction of the basement membrane surrounding the microvascular endothelial cells, which allows endothelial cells to extend cytoplasmic buds in the direction of chemotactic factors. Migrating endothelial cells elongate, divide and eventually form tube structures which join to form mature new capillaries. Results of in vitro experiments, in vivo experiments, and clinical studies suggest that peptide growth factors can play key regulatory roles in each step of neovascularisation through both direct and indirect actions. At sites of vascular injuries, degranulating platelets release PDGF, IGF-I, EGF, and TGF-beta. Macrophages and neutrophiles drawn into the ischaemic or injured areas synthesise and release TGF-alpha, TGF-beta, and PDGF, and wounded endothelial cells secrete FGF. These peptide growth factors can stimulate migration, mitosis and differentiation of endothelial cells in culture and can induce neovascularisation in animal models. Clinical correlations suggest that peptide growth factors in the vitreous such as IGF-I and bFGF may promote diabetic retinopathy. As the biological mechanisms of neovascular growth factors become better understood, it may be possible to develop therapeutic approaches to selectively inhibit the peptide growth factors which regulate neovascular diseases.

Angiogenesis Inducing Agents↗
PubMed1991

Plasminogen activator production by human retinal endothelial cells of nondiabetic and diabetic origin.

The authors examined the effect of insulin-like growth factor 1 (IGF 1), epidermal growth factor (EGF), and acidic fibroblast growth factor (AFGF) on the synthesis by human retinal endothelial cell (HREC) of plasminogen activators (PA; tissue-type [t-PA] and urokinase-type [u-PA]) and plasminogen activator inhibitor (PAI). Immunologic and functional assays for t-PA, u-PA, and PA1 were conducted with cell lines derived from three diabetics and three nondiabetic controls. Confluent HREC of nondiabetic origin did not respond to IGF I (100 ng/ml) with any change of t-PA antigen in the medium (10.7 +/- 1.1 ng/ml unstimulated versus 10.1 +/- 0.8 ng/ml) stimulated, P = not significant). Likewise AFGF and EGF caused no significant change of t-PA levels. Both IGF I and EGF caused a significant increase of t-PA from HREC of diabetic origin (9.6 +/- 0.8 ng/ml unstimulated versus 16.6 +/- 1.9 ng/ml IGF I-stimulated, P less than 0.001, and 14.6 +/- 2.7 ng/ml EGF-stimulated P less than 0.005). Supplementation of AFGF had no effect on HREC of diabetic origin. In confluent cultures, only small quantities of u-PA were detected. After wounding confluent cultures, u-PA activity was associated with cells migrating from the wound edges. Functional PA activity was also measured by chromogenic assay. Results further supported a predominance of t-PA activity being produced by confluent HREC in culture. These results suggest that modulation of PA production by HREC is influenced by exposure to growth factors, by the state of confluency, and the origin of the cells (diabetic vesus nondiabetic).

Aged↗
PubMed1991

Measurement of gene expression in human retinal microvessels by solution hybridization.

Changes in gene expression could play a central role in the phenotypic abnormalities of the retinal vascular cells observed in diabetic retinopathy and other retinal diseases. To measure gene expression in human retinal microvessels, a RNA-probe excess solution hybridization assay was used. Retinal microvessels were isolated from eyes obtained within 36 hr of death, and intact RNA was extracted by the guanidine method. Hybridization of poly(A)+ RNA northern blots revealed only the cytoskeletal beta-actin message; by using the more sensitive solution hybridization assay, the plasminogen activator-inhibitor 1 (PAI-1) and von Willebrand factor (vWF) mRNAs were quantified. The prevalence of these transcripts in the retinal microvessels was 0.04 x 10(6) copies/ng RNA for PAI-1 and 0.14 x 10(6) copies/ng RNA for vWF, much less than the prevalence in human umbilical vein endothelial cells (1.93 x 10(6) and 3.90 x 10(6), respectively). The PAI-1 mRNA levels in retinal microvessels isolated from five type II diabetic patients were significantly higher than those in vessels isolated from ten age-matched controls (0.06 x 10(6) versus 0.04 x 10(6) copies/ng RNA, P less than 0.05). The solution hybridization assay accurately measured low-abundance mRNAs in human retinal microvessels; determination of gene expression in these cells could aid in understanding the pathogenesis of important ophthalmologic diseases such as diabetic retinopathy.

Actins↗
PubMed1991

Eye care guidelines for patients with diabetes mellitus.

Diabetic retinopathy is the leading cause of blindness in adults under age 65 in the United States. Risk is related to the stage of retinopathy. With new treatments available, it is imperative that diabetics be properly screened. Those with retinopathy of more than five years duration should be referred to an ophthalmologist. Since photocoagulation has been shown effective for treatment of macular edema and proliferative retinopathy, it is imperative that these patients be referred for timely evaluation.

Diabetes Mellitus↗
PubMed1990

Insulin-like growth factor I stimulates proliferation, migration, and plasminogen activator release by human retinal pigment epithelial cells.

The migration of retinal pigment epithelial (RPE) cells from their normal anatomic position to a new position in the vitreous cavity is a critical feature of proliferative vitreous retinopathy. To determine if insulin-like growth factor I (IGF I), which is present in the vitreous fluid of diabetics, stimulates RPE cells, we examined the effects of IGF I on the proliferation, chemotaxis, and release of plasminogen activator by these cells. At the concentrations of IGF I tested, significant proliferation of RPE cells is seen. Significant chemotaxis of the RPE cells also is seen at all the concentrations of IGF I tested. The mean number of migrating cells per high-powered field in control studies was 43 +/- 13 (x +/- SEM), and for IGF I at 2.5 ng and 50 ng/ml the mean numbers of migrating cells were 96 +/- 17 and 483 +/- 62, respectively (P less than 0.001 for each comparison). The IGF I response was noted to be dose-dependent. The chemotactic response noted at 50 ng/ml of IGF I was greater than the positive chemotactic control of 10% fetal calf serum. Addition of alpha IR-3, an IGF I receptor antibody, eliminated the IGF I chemotactic response. The effect of IGF I on the secretion of plasminogen activators was assessed using an immunological assay for tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI). Media conditioned by RPE cells have measureable levels of PAI and t-PA antigen. IGF I supplementation resulted in an increase of t-PA secretion and PAI secretion over basal levels. These studies support a role for IGF I in modulating RPE cell function.

Antibodies, Monoclonal↗
PubMed1989

Insulinlike growth factors in patients with active nephrotic syndrome.

The serum concentrations of insulinlike growth factors 1 and 2 (IGF-1 and IGF-2) were measured by specific radioimmunoassays in 25 nephrotic patients. The serum concentration of IGF-1 in nephrotic patients (mean +/- SEM, 169 +/- 17 ng/mL) was significantly lower than that observed in 20 control subjects matched for sex and age (338 +/- 36 ng/mL). The IGF-2 serum concentration was significantly lower in nephrotic patients (343 +/- 22 ng/mL) than in control subjects (898 +/- 80 ng/mL). The IGF-1 and IGF-2 150-kd and 45-kd carrier protein complexes were found in the urine of nephrotic patients, whereas there was no binding of radiolabeled IGF-1 or IGF-2 to IGF carrier proteins in the urine of control subjects. The low serum IGF-1 and IGF-2 levels observed in nephrotic patients could be partially due to the increased urinary losses of the IGF carrier proteins.

Adolescent↗
PubMed1989

Fibrinolytic capacity following stimulation with desmopressin acetate in patients with diabetes mellitus.

Tissue plasminogen activator (t-PA), tissue plasminogen activator inhibitor, (PAI), and von Willebrand factor (vWF) were measured in 30 diabetics and 17 control subjects. These studies were performed to clarify the role of obesity in causing abnormalities of the fibrinolytic system in diabetics. The t-PA antigen response measured after the infusion of desmopressin acetate (DDAVP) was similar in all groups. Peak responses to DDAVP for controls, type I diabetics, and type II diabetics were 21.2 +/- 9.5 ng/mL, 27.5 +/- 9.0 ng/mL, and 28.8 +/- 11.0 ng/mL (NS), respectively. These responses did not correlate with the body mass index (BMI) or any other of the indices examined. A significant decrease of t-PA activity as contrasted with t-PA antigen following DDAVP occurred in type II diabetics only. The decrease of t-PA activity strongly correlated with greater basal levels of plasminogen activator inhibitor in these same subjects. The plasma level of plasminogen activator inhibitor correlated with BMI but with no other index examined. In contrast to t-PA activity and PAI, vWF responses to DDAVP inversely correlated to basal vWF concentration in all groups. Basal concentrations of vWF were increased in both type I and II diabetics and showed no relationship to degree of obesity. In summary, these results suggest that type II diabetic subjects have decreased t-PA activity, which is best explained by increased levels of PAI. The increased PAI appears related to obesity and not diabetes per se.

Adolescent↗
PubMed1989

Insulin-like growth factor secretion by human B-lymphocytes: a comparison of cells from normal and pygmy subjects.

Freshly isolated human B-lymphocytes from eight subjects and Epstein-Barr virus-transformed human B-lymphocytes from seven subjects were examined for their capacity to secrete insulin-like growth factor-I (IGF-I) and IGF-II and for their capacity to respond to human GH. Similar studies were conducted with Epstein-Barr virus-transformed lymphocytes collected from six African pygmies. When transformed B-lymphocytes from normal stature subjects were cultured for 3 weeks in RPMI-1640 medium (6 x 10(3) cells/75-cm2 flask at seeding), significant amounts of IGF-I, but no IGF-II, were produced. GH (150 ng/mL) significantly increased for control cells the amount of IGF-I produced at each sampling interval compared to that by unstimulated cultures (P less than 0.05 at 1 week; P = 0.005 at 3 weeks). At 3 weeks, cell counts of cultures compared were 4.13 +/- 0.39 X 10(6)/mL for unstimulated cells and 4.23 +/- 0.87 X 10(6)/mL for GH-stimulated cells. IGF-I production at this time interval by unstimulated cells was 2.8 +/- 2.3 ng/mL, and that by GH-stimulated cells was 12.3 +/- 2.5 ng/mL (P = 0.005). Cell multiplication rates of control cultures were increased in 1 week by GH stimulation [GH stimulated, [16.7 +/- 22.0 X 10(4) cells, unstimulated, 5.73 +/- 4.1 X 10(4) cells; (mean +/- SD); n = 14; P less than 0.01]. Similar results occurred with GH studied at a lower concentration of 10 ng/mL for 3 weeks. Freshly isolated B-lymphocytes did not secrete IGF-I and II after 5 days of culture with GH. Cultures established from cells derived from pygmies produced significantly less IGF-I (4.24 +/- 2.62 ng/mL) when stimulated with 150 ng/mL GH than cultures of cells from normal stature subjects (12.3 +/- 2.5 ng/mL; 0.005 less than P less than 0.01). The cultures compared had a similar cell density. A similar significant difference in IGF-I secretion occurred between cultures of pygmy and control cells stimulated with 10 ng/mL GH. These data are consistent with previous in vivo studies in which pygmies failed to increase IGF-I and exhibit metabolic responses to exogenous GH.

Adult↗