Search PubMed⌕ Search

Biomedical subjects

M Lorenzi

Publications and source records attributed to M Lorenzi.

At least 37 records · Page 2Linked to original sources
PubMed1998

Expression of vascular endothelial growth factor in the human retina and in nonproliferative diabetic retinopathy.

Vascular endothelial growth factor (VEGF)/vascular permeability factor is a likely angiogenic mediator in proliferative diabetic retinopathy, and its role is under scrutiny in the pathogenesis of the capillary leakage characteristic of background diabetic retinopathy. To examine whether the diabetic milieu induces or increases retinal VEGF expression in humans, we examined retinas from nondiabetic eye donors and donors with 9 +/- 5 years of diabetes and documented microangiopathy. To identify possible confounding effects of the postmortem period, we also studied the postmortem stability of the VEGF transcript and the expression of the VEGF protein in rat retinas. In both human and rat retina we detected by Northern analysis a 4.2-kb VEGF mRNA species and by reverse transcriptase polymerase chain reaction the transcripts encoding VEGF165 (the most abundant), VEGF121, and VEGF189. By in situ hybridization and immunohistochemistry VEGF mRNA and protein co-localized at the ganglion cell, inner nuclear, and outer plexiform layers and in the walls of the blood vessels (where mRNA was scarce). The protein was additionally detected in photoreceptors. The abundance and distribution of VEGF mRNA and protein were not altered in the diabetic retinas, indicating that the diabetic environment is not sufficient to increase retinal VEGF expression. The demonstration that VEGF is constitutively expressed in the adult retina and is localized to discrete neural cells and their processes proposes a role for the cytokine in retinal homeostasis and/or function.

Aged↗
PubMed1997

Cytoskeletal changes induced by excess extracellular matrix impair endothelial cell replication.

Thickening of basement membranes is an early and characteristic feature of diabetic vessels, but its consequences on the properties of vascular cells remain undefined. We investigated whether and how excess extracellular matrix (ECM) alters the replication of vascular endothelial cells in vitro. To test the effects of endogenous excess matrix, human umbilical vein endothelial cells (HUVEC) were plated on ECM produced under culture conditions (high ambient glucose) that increase ECM synthesis. Four of six HUVEC isolates plated on such ECM yielded a lower cell number (68 +/- 18%) than cells plated on control ECM. Growth inhibition was observed in HUVEC cultured on elevated concentrations (10 and 50 microg/ml) of exogenous fibronectin, when compared with HUVEC plated on tissue culture plastic or 0.25, 1.0, and 5.0 microg/ml fibronectin; the decreased replication was attributable to delayed transit through the G1 phase of the cell cycle. HUVEC grown on both 1 and 10 microg/ml fibronectin exhibited a modest upregulation of the fibronectin-specific integrin receptor alpha5beta1, and increased attachment to fibronectin substratum. However, unique to the HUVEC plated on growth-inhibitory concentrations of fibronectin was a redistribution in situ of integrins and vinculin to form more numerous focal adhesions, and an increased polymerization of cytoskeletal actin to form stress fibers. Concentrations (0.01 microg/ml) of cytochalasin D intended to prevent excess actin polymerization prevented the growth inhibition. Thus, excess ECM hampers endothelial cell replication in vitro through increased cell-ECM adhesion and attendant cytoskeletal rearrangements. These phenotypic changes provide probes to test whether cell-ECM interactions are altered in diabetic vessels in a direction that may compromise orderly endothelial cell renewal and its antithrombogenic function.

Actins↗
PubMed1997

Mesangial cell abnormalities in spontaneously hypertensive rats before the onset of hypertension.

To identify kidney biosynthetic abnormalities that may precede the onset of hypertension, we studied the expression of fibronectin (FN) and collagen IV (Coll IV) in young SHR (4 weeks of age) whose systolic blood pressure was normal and similar to that of age-matched control WKY rats. In isolated glomeruli the level of FN protein assessed by immunoblotting tended to be lower in the SHR than in the WKY rats. By Northern analysis the FN/actin mRNA ratio was significantly lower in glomeruli from SHR (0.56 +/- 0.47) than in glomeruli from WKY rats (2.0 +/- 0.8). These abnormalities were maintained in vitro since the expression of FN was significantly lower in SHR than in WKY cultured mesangial cells (FN/actin mRNA ratio = 0.84 +/- 0.46 vs. 1.9 +/- 0.7, P = 0.029). No differences in Coll IV mRNA or protein levels were observed in SHR glomeruli and mesangial cells when compared with WKY rats. The levels of aortic FN and Coll IV mRNAs were not different in SHR and WKY rats. In addition, mesangial cells from SHR showed a significantly higher growth rate than those from WKY. The biosynthetic and proliferative abnormalities observed in the SHR mesangial cells appear to reflect genetic characteristics, and could provide novel insights into cellular mechanisms linking the genetics of hypertension with predisposition to glomerular pathology.

Actins↗
PubMed1997

Visual and verbal short-term memory deficits in childhood leukemia survivors after intrathecal chemotherapy.

Assessed survivors of childhood lymphoblastic leukemia (ALL) treated with intrathecal chemotherapy, using the Wide Range Assessment of Memory and Learning (WRAML), compared to controls without cancer, matched as closely as possible in age, SES, and gender. Mild, but consistent, deficits were found in both visual-spatial and verbal single-trial memory tasks. In multitrial learning, only visual-spatial tasks resulted in deficient scores, while verbal learning was within the normal range. IQ results indicated scores 10-20 points lower in the ALL group. Memory results are related to deficits in strategic planning and attentional distractiveness. The WRAML may be a useful clinical tool to evaluate differential memory deficits in children with ALL.

Adolescent↗
PubMed1996

Accelerated death of retinal microvascular cells in human and experimental diabetic retinopathy.

To reconstruct the mechanisms for the vasoobliteration that transforms diabetic retinopathy into an ischemic retinopathy, we compared the occurrence of cell death in situ in retinal microvessels of diabetic and nondiabetic individuals. Trypsin digests and sections prepared from the retinas of seven patients (age 67 +/- 7 yr) with .9 +/- 4 yr of diabetes and eight age- and sex-matched nondiabetic controls were studied with the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) reaction which detects preferentially apoptotic DNA fragmentation. The count of total TUNEL+ nuclei was significantly greater in the microvessels of diabetic (13 +/- 12 per one-sixth of retina) than control subjects (1.3 +/- 1.4, P = 0.0016), as were the counts of TUNEL+ pericytes and endothelial cells (P < 0.006). The neural retinas from both diabetic and nondiabetic subjects were uniformly TUNEL-. Retinal microvessels of rats with short duration of experimental diabetes or galactosemia and absent or minimal morphological changes of retinopathy, showed TUNEL+ pericytes and endothelial cells, which were absent in control rats. These findings indicate that (a) diabetes and galactosemia lead to accelerated death in situ of both retinal pericytes and endothelial cells; (b) the event is specific for vascular cells; (c) it precedes histological evidence of retinopathy; and (d) it can be induced by isolated hyperhexosemia. A cycle of accelerated death and renewal of endothelial cells may contribute to vascular architectural changes and, upon exhaustion of replicative life span, to capillary obliteration.

Aged↗
PubMed1996

Early biosynthetic changes in the diabetic-like retinopathy of galactose-fed rats.

The histological lesions of diabetic micro-angiopathy have a long latency, but vascular cell function may be affected at early stages of the process. Rats with experimental galactosaemia develop a diabetic-like retinopathy in the absence of other metabolic abnormalities characteristic of diabetes mellitus; basement membrane thickening is measurable in their retinal vessels after 7 months of galactose feeding. To examine the course of biosynthetic changes relevant to the process, retinal expression of collagen IV and fibronectin were compared in rats fed a 30% galactose diet or a control diet for 5 or 9 weeks. Total retinal RNA was studied by reverse transcription-polymerase chain reaction; the fibronectin primers encompassed the alternatively spliced EIIIA exon. The levels of alpha 1 (IV) collagen and fibronectin mRNAs were measured relative to an internal standard (beta-actin mRNA). The proportion of EIIIA+ to EIIIA- fibronectin transcripts was similar in the retinas of control and galactose-fed rats, which, however, showed increased levels of both fibronectin and collagen IV mRNAs in the presence of unchanged beta-actin mRNA levels. An upward trend was detected by 5 weeks of galactose feeding; and after 9 weeks the fibronectin/actin ratio was 1.2 +/- 0.3 vs 0.8 +/- 0.2 in controls (p = 0.015) and the collagen IV/actin ratio was 1.3 +/- 0.3 vs 0.9 +/- 0.2 in controls (p = 0.04). Thus, hyperhexosaemia of a few weeks' duration is a perturbation sufficient to increase the synthesis of basement membrane components in the retina. The search for additional early biosynthetic changes should assist in reconstructing the pathogenesis of hexose-induced retinal microangiopathy.

Actins↗
PubMed1996

Fibronectin overexpression in retinal microvessels of patients with diabetes.

PURPOSE: To examine whether fibronectin is synthesized in the adult human retina and whether diabetes alters its amount, level of expression, and pattern of splicing. METHODS: Retinas were obtained after death from 24 patients (age, 64 +/- 6 years) with 8 +/- 5 years of diabetes and 24 age-matched control subjects. Retinal localization of fibronectin and its amount in retinal vessels were examined with the immunoperoxidase technique. Fibronectin mRNA in the total retina was studied with reverse transcriptase-polymerase chain reaction using primers encompassing the alternatively spliced exon ED-A. The levels of fibronectin mRNA were measured relative to an internal standard (beta-actin mRNA). To detect and measure fibronectin expression in microvessels, retinal trypsin digests were studied by in situ hybridization. RESULTS: In the adult human retina, fibronectin is present exclusively in the walls of the vessels, including capillaries, and in the internal limiting membrane. In diabetic vessels, immunoreactive fibronectin was increased 1.4-fold over levels in control vessels (P = 0.05). Fibronectin is synthesized locally, and the predominant mRNA species excludes the ED-A region. The splicing pattern was not altered by diabetes. However, in the retinas of patients with diabetes, the levels of fibronectin mRNA were increased 1.3-fold over levels in control retinas (P = 0.028), whereas actin mRNA levels were similar in the two groups. Levels of fibronectin mRNA showed a substantial increase in the microvessels of patients with diabetes (0.49 +/- 0.18 grains/cell versus 0.15 +/- 0.12 grains/cell in control microvessels; P = 0.009. CONCLUSIONS: Fibronectin is synthesized in the adult human retina. Diabetes increases the amount of fibronectin in retinal vessels and upregulates its expression without changing the splicing pattern of the ED-A segment. Increased synthesis and deposition of fibronectin by microvascular cells may modify the interactions of cells and matrix with functional consequences relevant to the lesions of retinopathy.

Aged↗
PubMed1995

Activity and expression of the Na+/H+ exchanger in human endothelial cells cultured in high glucose.

Establishing whether high ambient glucose affects the plasma membrane Na+/H+ exchanger is relevant to understanding the adverse effects of high glucose on cell replication and the mechanisms of the increased exchanger activity encountered in diabetic patients with nephropathy. In 8 primary and 15 first-passage isolates of human endothelial cells cultured in 30 mmol/l glucose for 8.7 +/- 2.3 and 15.8 +/- 2.3 days, respectively, we determined Na+/H+ exchanger activity and mRNA levels. Activity was determined by measuring 22Na+ influx in the presence or absence of dimethylamiloride (DMA) after intracellular acidification. We also measured fibronectin mRNA because fibronectin provides signals for cell replication through the Na+/H+ antiporter. Control cells grown in 5 mmol/l glucose showed at morphologic confluency a total Na+ influx (in nmol.mg protein-1.min-1) of 10.1 +/- 3.2 in primary and 11.7 +/- 2.2 in first subculture, which was reduced to 5.3 +/- 0.3 in the presence of DMA. Paired cultures exposed to 30 mmol/l glucose and exhibiting pHi and cell densities identical to controls showed in both primary and first subculture a reduction in total Na+ influx (delta = -0.98 +/- 0.93 nmol.mg protein-1.min-1 p < 0.005) whereas DMA-resistant Na+ influx was identical to that of control. Neither chronic hypertonicity nor acute exposure to high glucose mimicked the effects of chronic high glucose. The level of the Na+/H+ exchanger isoform 1 (NHE-1) mRNA was unchanged by high glucose whereas fibronectin mRNA levels were increased 1.5-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗
PubMed1995

Treatment of advanced colorectal cancer with high-dose intensity folinic acid and 5-fluorouracil plus supportive care.

This randomised clinical trial, involving patients with advanced colorectal cancer, was carried out to compare the effectiveness of accelerated folinic acid (FA) plus 5-fluorouracil (5-FU) with that of the conventional regimen of 5-FU alone. Both regimens were administered with simulataneous supportive care. 185 patients were eligible: 94 were randomly allocated to receive FA 200 mg/m2 i.v. plus 5-FU 400 mg/m2 i.v. on days 1-5 every 3 weeks; and 91 to receive 5-FU 400 mg/m2 i.v. on days 1-5 every 4 weeks. The response rate was 33.3% in the accelerated FA/5-FU and 18.6% in the 5-FU arm (P = 0.045). Median survival was 13.5 months in the FA/5-FU arm and 7.5 months in the 5-FU arm (P = 0.039). Toxicity was mild and slightly more pronounced in the FA/5-FU arm (P = 0.078). This study indicates that, in patients with advanced colorectal cancer, accelerated chemotherapy with FA and 5-FU and simultaneous supportive care is capable of achieving a higher response rate and longer survival than conventional 5-FU alone, without severe toxicity.

Adult↗
PubMed1995

The effects of high glucose on human endothelial cell growth and gene expression are not mediated by transforming growth factor-beta.

BACKGROUND: Because accumulation of extracellular matrix is a prominent characteristic of the microangiopathy that complicates long-term diabetes, a pathogenetic role for transforming growth factor beta (TGF-beta) is being considered. Having observed that glucose levels mimicking diabetic hyperglycemia induce in vitro endothelial cell overexpression of extracellular matrix molecules, decreased replication, and increased levels of TGF-beta mRNA, we have examined whether the effects of high glucose are mediated by autocrine TGF-beta. EXPERIMENTAL DESIGN: TGF-beta levels were measured by bioassay in the media conditioned by human umbilical vein endothelial cells cultured in the presence of high (30 mM) or normal (5 mM) glucose concentrations. The effect of high glucose was tested on the proliferation of two epithelial cell lines, one (Mv1Lu) exquisitely sensitive to TGF-beta and the other (DR mutants) insensitive to the cytokine. To examine whether high glucose and TGF-beta affect cellular programs in a similar manner, the effects of high glucose and exogenous TGF-beta were compared on proliferation and gene expression of endothelial cells. RESULTS: Media conditioned by endothelial cells cultured in high or normal glucose contained similar amounts of TGF-beta (4.9 +/- 3.5 and 3.7 +/- 2.5 ng/10(6) cells, respectively (mean +/- SD)), all in the latent form. The replication of parental Mv1Lu cells and their DR mutants was decreased by high glucose to the same extent. Whereas the inhibitory effect of high glucose on endothelial cell replication was reversible, that of TGF-beta was not. Both perturbations induced up-regulation of fibronectin expression, but the effects were additive. Only TGF-beta induced overexpression of Type IV collagenase. CONCLUSIONS: These combined observations indicate that (a) endothelial cells exposed to high glucose do not secrete TGF-beta in excess of control cells, (b) there are growth-inhibitory effects of high glucose that are independent of TGF-beta, and (c) high glucose and TGF-beta exert their effects through distinct pathways and at different loci.

Cell Division↗
PubMed1994

Increased expression of basement membrane collagen in human diabetic retinopathy.

Basement membrane thickening is the most prominent and characteristic feature of early diabetic microangiopathy. Unknown is not only the causative process but also whether the thickening reflects increased synthesis of specific components. Because collagen type IV is uniquely present in basement membranes and represents their predominant structural element, we studied its expression in retinas obtained postmortem from five patients with 8 +/- 3 yr of diabetes and six nondiabetic controls. The collagen IV transcript proved to be rare in adult human retina and undetectable by Northern analysis. We thus identified a set of primers and conditions to detect the transcript by the reverse transcriptase polymerase chain reaction and to measure its level relative to an endogenous internal standard (beta-actin mRNA). In the diabetic patients the levels of collagen IV mRNA were increased twofold over levels in controls, whereas the actin mRNA levels were similar in the two groups. Hence, the collagen IV/actin ratio was 0.53 +/- 0.15 in diabetic samples and 0.24 +/- 0.09 in control samples (P = 0.004). These results indicate that diabetes induces a twofold increase in the expression of collagen IV by the cells that synthesize basement membranes in the adult retina (vascular cells). Insofar as high ambient glucose in vitro elicits the same effect, it may be proposed that basement membrane thickening in diabetes results from enhanced synthesis of specialized component molecules sustained by hyperglycemia.

Actins↗
PubMed1994

Vascular wall von Willebrand factor in human diabetic retinopathy.

PURPOSE: To reconstruct the role played by vascular endothelium in the elevation of circulating von Willebrand factor (vWf) in diabetic patients with microangiopathy and, specifically, to determine whether storage and synthesis of vWf is altered in diabetic retinal vessels. METHODS: Trypsin digests were prepared from retinas obtained post mortem from 11 patients (age 62 +/- 9 years, mean +/- SD) with 9 +/- 5 years of diabetes and 12 nondiabetic control subjects matched for age and sex. Trypsin digests were inspected for the presence of lesions of diabetic retinopathy; vWf protein was localized by indirect immunofluorescence; and vWf mRNA levels were studied by in situ hybridization. RESULTS: vWf immunofluorescence was present in vessels of all sizes. The granular fluorescence was localized to the endothelial cell cytoplasm. Pattern and intensity of staining in diabetic microvessels and large vessels were similar to those observed in the vessels of nondiabetic subjects. The amount of vWf mRNA detected by in situ hybridization in retinal endothelial cells was similar in diabetic (0.92 +/- 0.32 grains/cell) and control (0.91 +/- 0.42 grains/cell) microvessels. Likewise, no differences were observed in vWf mRNA levels in the large vessels of diabetic (0.073 +/- 0.034% grain area) and control (0.069 +/- 0.018 grain area) subjects. CONCLUSIONS: These observations are compatible with the occurrence in diabetes of the slow release of endothelial vWf through the pathway of vWf secretion not linked to synthesis, ie, the regulated pathway.

Adult↗
PubMed1993

Integrin overexpression induced by high glucose and by human diabetes: potential pathway to cell dysfunction in diabetic microangiopathy.

The nature of the process leading to the acellular nonperfused capillaries of diabetic microangiopathy remains unknown. Because these capillaries manifest thickened basement membranes, we asked whether the process causing deposition of excess extracellular matrix in diabetes modifies cell-matrix interactions in a direction that would compromise cell renewal. In 44 individual isolates of human umbilical vein endothelial cells we observed that high glucose concentrations (30 mM) induce coordinate increases in the levels of mRNAs encoding fibronectin and the fibronectin-specific integrin receptor alpha 5 beta 1 as well as in the cognate proteins. Expression of the integrin subunit alpha 3, component of the alpha 3 beta 1 polyspecific receptor for fibronectin, laminin, and collagen, was also up-regulated by high glucose. Overexpression of integrins correlated with increased cell attachment to exogenous fibronectin and laminin as well as to complex matrix. Moreover, cells exhibited firmer steady-state adhesion to their own matrix. To correlate these in vitro observations with events in human diabetic retinopathy we measured integrin levels in retinal trypsin digests prepared from 10 patients with 8.2 +/- 1.6 (mean +/- SE) years of diabetes and 10 age- and sex-matched nondiabetic controls. Microvessels of diabetic patients showed increased immunostaining for beta 1 integrin (P = 0.025) when compared with control microvessels. These data show that high glucose and diabetes increase integrin expression and thus alter the interaction of vascular endothelial cells with their basement membranes in the direction of firmer cell-matrix adhesion. This could compromise the migration and replication critical to the reendothelialization process and contribute to microvascular occlusion.

Cell Adhesion↗
PubMed1993

Nitrogen metabolism during liver regeneration.

Nitrogen metabolism was investigated in regenerating liver-bearing rats through the following parameters: (1) liver aminoacid content, (2) plasma and urinary urea and creatinine, (3) plasma and urinary oxypurines, uric acid and allantoin. Two groups of aminoacids were considered: (1) the essential aminoacids (phenylalanine, tyrosine, isoleucine, lysine, leucine, valine, arginine, histidine and methionine); (2) the non-essential aminoacids (aspartic acid, asparagine, glutamic acid, glutamine, alanine, glycine, serine, threonine and proline). Some of the first group tended to decrease, and those of the second group to increase, immediately after partial hepatectomy. Few ketogenic aminoacids are probably oxidized to provide energy. The flux of aminoacids for gluconeogenesis is minutely controlled, therefore, those of the second group being spared at first and set aside for protein synthesis, which increases on the second and third days after partial hepatectomy. Plasma and urinary urea, oxypurines, uric acid and allantoin did not show any significant variations after partial hepatectomy. The conclusion emerging from the present research is that, although variations in aminoacid composition and metabolism and in purine nucleotide metabolism have been demonstrated to occur in the regenerating liver, the overall nitrogen catabolism, as reflected by the principal end products, does not undergo substantial variations. The remaining liver is able to fulfil this function.

Allantoin↗
PubMed1993

Maternal diabetes induces increased expression of extracellular matrix components in rat embryos.

The mechanisms responsible for the increased incidence of congenital malformations in offspring of diabetic mothers are poorly understood. Because the abnormal metabolic milieu of diabetes induces in the adult organism increased synthesis of basement membrane components, and these molecules play a prominent role in morphogenesis, we investigated whether maternal diabetes or high glucose levels disturb extracellular matrix synthesis in rat embryos. In gestational day 11 embryos, maternal diabetes induced a small but significant increase in laminin B1 (127 +/- 40% of control, mean +/- SD, P < 0.02) but not in fibronectin mRNA (101 +/- 26% of control). Day 12 embryos from diabetic mothers showed a larger increment in laminin B1 (179 +/- 91% of control, P < 0.02) and also an increase in fibronectin mRNA (172 +/- 73% of control, P < 0.02). A similar increase in the expression of fibronectin was observed in the kidneys and hearts of day 20 fetuses dissected from diabetic rats. High glucose levels mimicked in vitro the effects of maternal diabetes. Day 9 embryos cultured for 48 h in 50 mM D-glucose showed, akin to the day 11 embryos in vivo, an increase in laminin B1 mRNA (129 +/- 47% of control) and no changes in fibronectin mRNA (106 +/- 35% of control). The finding that maternal diabetes induces increased expression of extracellular matrix components in developing embryos establishes a link with the abnormalities occurring in the chronic complications of diabetes and proposes a new path of investigation for the mechanism of teratogenicity of the diabetic milieu.

Animals↗
PubMed1992

Increased expression of tissue plasminogen activator and its inhibitor and reduced fibrinolytic potential of human endothelial cells cultured in elevated glucose.

In diabetic patients, elevated plasma levels of t-PA and PAI-1 accompany impaired fibrinolysis. To identify mechanisms for these abnormalities, we examined whether vascular endothelial cells exposed to high glucose upregulate t-PA and PAI-1 production and whether ambient PA activity is decreased concomitantly. In 17 cultures of human umbilical vein endothelial cells grown to confluency in 30 mM glucose, the t-PA antigen released to the medium in 24 h was (median) 52 ng/10(6) cells (range 10-384) and the PAI-1 antigen was 872 ng/10(6) cells (range 217-2074)--both greater (P less than 0.02) than the amounts released by paired control cultures grown in 5 mM glucose--29 ng/10(6) cells (range 7.5-216) and 461 ng/10(6) cells (range 230-3215), respectively. In the presence of high glucose, the steady-state levels of t-PA and PAI-1 mRNAs were increased correspondingly (median 142 and 183% of control, respectively, P less than 0.05); high glucose per se and hypertonicity contributed to the upregulation in additive fashion. The PA activity of conditioned medium from cultures exposed to high glucose was 0.4 IU/ml (range 0.2-0.6), which was significantly lower (P less than 0.02) than the PA activity of control medium (0.5 IU/ml, range 0.2-0.9). No difference was observed when comparing the PA activities of acidified conditioned media, expected to be depleted of inhibitors. Thus, high glucose coordinately upregulates endothelial t-PA and PAI-1 expression through effects exerted at the pretranslational level and enhanced by even mild degrees of hypertonicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗