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*
Renal Unit, Ghent University, Belgium
Department of Pharmacology, Texas Biotechnology Corporation, Houston,
Texas.
Correspondence to Dr. An De Vriese, Renal Unit, University Hospital, OK12, De Pintelaan 185, B-9000 Ghent, Belgium. Phone: +32-9-2405301; Fax: +32-9-2404599; E-mail: an.devriese{at}rug.ac.be
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two widely known characteristics of early diabetic microvascular dysfunction are hyperpermeability to macromolecules and neovascularization (6,7). Abnormal passage of plasma proteins across the endothelium and their deposition in the basement membrane has been suggested to contribute to the basement membrane thickening typical of diabetic microangiopathy, although other mechanisms, including increased synthesis and decreased degradation of matrix proteins, may play a role as well (8). Whereas hyperglycemia is recognized as pivotal in the development of these microvascular complications, the nature of the pathogenetic link between high ambient glucose concentrations and microvascular alterations remains a matter of debate, attesting to the complexity of the processes involved rather than to a lack of candidate mechanisms.
Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a heparin-binding angiogenic growth factor that displays a high specificity for endothelial cells. VEGF plays a prominent role in physiologic and pathologic angiogenesis (9,10). In addition, VEGF dramatically increases endothelial cell permeability and is established as one of the most potent endothelial permeabilizing agents identified thus far (11). Other biologically relevant actions of VEGF include an increase in blood flow and vascular conductance (12) and modulation of leukocyte kinetics (9,10). There is compelling evidence that endothelium-derived nitric oxide acts as a downstream mediator for VEGF (12,13).
Considering the cardinal features of hyperglycemia-induced microvascular dysfunction, VEGF is an attractive candidate to provide a pathogenetic link between high glucose exposure and the development of microvascular hyperpermeability and neoangiogenesis. In vitro exposure to high glucose concentrations rapidly induces VEGF expression in several cell types (14,15,16). Increased VEGF expression has been demonstrated in the retina, kidney, and nerve fibers of humans and experimental animals with diabetes (17,18,19,20,21). Peritoneal generation of VEGF has been observed in patients treated with glucose-based dialysate solutions (22). Vascular dysfunction induced by topical application of elevated glucose levels in a granulation skin chamber was attenuated by administration of neutralizing VEGF antibodies (Ab) (23). Although these studies present circumstantial evidence for a pathogenetic role of VEGF in glucose-induced microvascular alterations, direct evidence is lacking.
The purpose of this study was to characterize the hyperglycemia-induced functional and structural alterations in the peritoneal microvasculature of the rat. In addition, a potential causative role for VEGF in these alterations was investigated by treating the experimental animals with a neutralizing monoclonal Ab specific for VEGF.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Anti-VEGF Ab Treatment
Monoclonal anti-VEGF Ab and isotype-matched control Ab were prepared as
described previously (23).
Briefly, female 8-wk-old BALB/c mice (Harlan Sprague Dawley, Inc.,
Indianapolis, IN) were immunized, then boosted 3 times, 21 d apart, by
intraperitoneal and subcutaneous injections of 50 µg of
rhVEGF165, emulsified with an equal volume of Complete Freund's
Adjuvant for the primary immunization and Incomplete Freund's Adjuvant for
secondary immunizations. The mouse with the highest serum titer to
rhVEGF165 as measured by enzyme-linked immunosorbent assay was
injected intravenously with an additional 30 µg of immunogen in
phosphate-buffered saline (PBS), 21 d after the last immunization. Three d
later, spleen cells were harvested for production of hybridomas to
rhVEGF165. The hybridoma cell line with the highest Ab titer and
neutralizing Ab activity was selected after cloning 3 to 4 times by limiting
dilution in 96-well microtiter plates, then grown in a Cellmax Bioreactor
(Spectrum, Rancho Dominguez, CA) using Dulbecco's modified Eagle's medium
culture media. Purified Ig was prepared by protein A chromatography. The
isotype and light chain composition of the Ab and the characterization of
neutralizing activity were performed as described previously
(23). One mg of the anti-VEGF
Ab or isotype-matched control Ab was injected intraperitoneally three times
per week, starting 2 d after induction of diabetes or the injection of citrate
buffer until the final experiments 6 wk later.
To determine the efficiency of the anti-VEGF treatment, we obtained serum samples at baseline and after 2, 4, and 6 wk of treatment with anti-VEGF or with control Ab. The samples were drawn from the tail vein just before the next Ab administration. Binding studies were performed using a fusion protein composed of the seven-loop ectodomain of fms-like tyrosine kinase fused to the heavy chains of a mouse IgG2a Ab. The fusion protein was captured onto Immulon 4 strip wells with IgG2a-specific goat anti-mouse Ab (Sigma, St. Louis, MO). Increasing concentrations of monoclonal anti-VEGF Ab (used as standards) and increasing dilutions of serum (1:50, 1:100, and 1:250) were incubated separately with 4 ng/ml 125I-VEGF (Biomedical Technologies Inc., Stoughton, MA) for 30 min at 37°C before adding to the receptor. Nonspecific binding was defined as the binding measured in the presence of a 100-fold molar excess of unlabeled VEGF. Incubations were terminated after 60 min at 22°C by washing the wells once with 400 µl of ice-cold PBS, then twice more with 200 µl of ice-cold PBS. Bound VEGF was measured in a gamma spectrometer (Life Technology Inc., Schaumburg, IL).
Peritoneal Transport Studies
Rats were anesthetized with thiobutabarbital (Inactin; RBI, Natick, MA; 100
mg/kg subcutaneously). The trachea was intubated, a jugular vein was
cannulated for continuous infusion of isotonic saline, and a carotid artery
was cannulated for blood sampling. The saline infusion rate was matched with
diuresis to maintain euvolemia. After 30 min, a silicon catheter (Venflon;
Becton Dickinson, Erembodegem-Aalst, Belgium) was inserted in the abdomen, and
15 ml of 3.86% glucose peritoneal dialysate solution (Dianeal; Baxter,
Nivelles, Belgium) was infused. Plasma and dialysate samples were collected at
t = 0, 30, 60, and 120 min for determination of creatinine, urea, and
glucose levels. Fructosamine and total protein levels were determined on the
first plasma sample only. Dialysate cultures were obtained at the end of the
experimental dwell, and animals were excluded from analysis if cultures were
positive. After 120 min, the abdomen was opened by midline incision for
collection of the dialysate fluid and for tissue sampling. The transport of
low molecular weight solutes was evaluated by calculating the mass transfer
area coefficient (MTAC) of urea and creatinine, using the Garred equation
(24):
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The initial peritoneal concentration of urea and creatinine is set at 0. The Garred formula is a simplified approach to calculate MTAC, assuming that the reflection coefficient of the solute is 0 and that the average solute concentration in the membrane equals the plasma concentration. The magnitude of the transport of small solutes is determined by the effective vascular surface area, which is dependent on the number of perfused peritoneal capillaries (25).
Intravital Microscopy
Rats were anesthetized with thiobutabarbital and cannulated as described
above. Cromoglycate (cromolyn sodium salt, 10 mg/kg intravenously; Sigma) was
administered 15 min before surgery, to block degranulation of mast cells
induced by the surgical manipulation. A small midline abdominal incision was
made, and a short segment of the small bowel was exteriorized, carefully
avoiding stretching. The visceral peritoneum was spread over a plexiglass
plate and superfused continuously with an isotonic, isocolloidal solution
(Hemaccel; Hoechst Marion Roussel, Marburg/Lahn, Germany) maintained at
37°C. The preparation was allowed to stabilize for 30 min after completion
of surgery. Observations were made with an Axiotech Vario 100 HD microscope
(Zeiss, Jena, Germany) using water immersion objectives (Achroplan 10x,
40x, 63x). The microscopic stage was driven by a stepping motor
control MCL-2 (Lang, Hüttenberg, Germany),
operated by a joystick or a software program (Wincommander;
Märzhäuser-Wetzlar,
Wetzlar, Germany) via an RS-232 interface. The tissue was transilluminated via
a fiberoptic using a light source (KL 1500; Schott, Wiesbaden, Germany)
equipped with a 150-W halogen lamp. Epifluorescence was performed with a
mercury lamp HBO 50 W and a FITC filter set (excitation filter BP 450 to 490,
dichroic mirror FT 510, emission filter LP 520). The resulting image was
displayed on a television monitor by a TK-1281 camera (Victor Company of Japan
LTD-JVC, Tokyo, Japan) and recorded by a video recorder (S-VHS Panasonic
AG-7355, Matsushita, Japan) for off-line analysis. All automatic gain controls
were switched off during the experiments. The video images were digitized with
an IP-8/AT Matrox image processing board and analyzed with image analysis
software (Cap-Image; Ingenieurbüro Zeintl,
Heidelberg, Germany) (26). The
technique allows the study of the peritoneal microcirculation, including small
arteries, arterioles, capillaries, postcapillary venules, venules, and small
veins (27). Larger vessels are
surrounded by fat and are not routinely visualized.
For the evaluation of macromolecular leakage, a venular segment with a diameter of 20 to 40 µm and an unbranched length of approximately 150 µm without any other vessels in the immediate vicinity was selected for study. FITC-bovine serum albumin (FITC-albumin, 50 mg/kg; Sigma) was administered as an intravenous bolus. When traumatic leaks were observed, the experiment was discontinued. Otherwise, epifluorescence recordings were made every 10 min for 120 min. On the digitized image of the venule under study, two intraluminal areas and two contiguous areas of perivenular interstitium were defined. The average gray scale value, ranging from 0 for black to 255 for white, was calculated for each area. As the molecule leaves the circulation, the intraluminal gray scale value falls and the perivascular gray scale value rises. Macromolecular leakage was defined as the ratio between the average gray scale value within the venule (Gv) and the average gray scale value in the perivenular interstitium (Gi) (28).
To evaluate microvascular density, we inspected all segments of the visceral peritoneum of the jejunum and ileum and the images were recorded. To avoid selection bias, we performed quantification in a predefined segment. The objective (10x) was positioned at random in the third segment of the distal ileum proximal from the caecum. With the aid of the Wincommander software, the microscopic stage was driven through a meander consisting of five steps of 1 mm in the X direction and five steps of 1 mm in the Y direction. The microscopic image was recorded at each of these 36 positions. The vessel length per area was determined for each microscopic image, and the average was calculated. Capillaries, defined as vessels with a luminal diameter <5 µm (27), were analyzed separately, and their relative contribution to total vessel length was calculated.
Statistical Analyses
The data are presented as mean ± SEM. ANOVA and unpaired t
tests were used as appropriate to test statistical significance. The
significance level was set at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In Vitro Serum VEGF-Inhibitory Activity
In the rats that were treated with anti-VEGF or control Ab, VEGF inhibitory
activities were measured at 0, 2, 4, and 6 wk of treatment, after dilution of
the serum at 1:50, 1:100, and 1:250. For the 1:50 dilution, baseline VEGF
inhibitory activity was low and not different between experimental groups
(Figure 1). An increased serum
inhibitory activity was found at 2, 4, and 6 wk of treatment with anti-VEGF Ab
in both diabetic and control rats as compared with baseline values and with
diabetic rats that were treated with control Ab. At 4 and 6 wk, inhibitory
activity was somewhat lower in anti-VEGF Ab-treated control rats as compared
with anti-VEGF Ab-treated diabetic rats. Because the inhibitory activity of
undiluted serum of anti-VEGF Ab-treated rats is almost 100%, the clinical
relevance of this difference may be minimal. As expected, the inhibitory
activities at 1:100 and 1:250 dilution of the serum were lower than those at
1:50 dilution but followed a similar pattern (data not shown).
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Peritoneal Transport Studies
Small solute transport was higher in 6-wk diabetic rats than in age-matched
controls, indicating the presence of a larger effective vascular surface area
in the diabetic peritoneum (Figure
2). The increase in effective vascular surface area was prevented
by treatment with anti-VEGF Ab but not by control Ab. Administration of
anti-VEGF Ab did not significantly affect the MTAC urea and creatinine in
control rats.
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Intravital Microscopy: Leakage of FITC-Albumin
The leakage of FITC-albumin was quantified as the change of
Gv/Gi during the observation period. Macromolecular
leakage was elevated in the 6-wk untreated and control Ab-treated diabetic
rats (Figure 3). The
hyperpermeability for albumin was prevented by treatment with anti-VEGF Ab.
After 90 min of observation, however, macromolecular leak was somewhat more
pronounced in anti-VEGF Ab-treated diabetic rats than in untreated controls
and in anti-VEGF Ab-treated controls but was still significantly lower than in
untreated diabetic and control Ab-treated diabetic rats. VEGF blockade did not
affect the permeability for FITC-albumin in control rats.
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Intravital Microscopy: Vascular Density
Intravital microscopy of the visceral peritoneum of control rats revealed a
progressively increasing microvascular density from the proximal jejunum
toward the distal ileum. In untreated and control Ab-treated diabetic rats,
this gradient was not present; irregularly arranged and dense vascular
networks were visible in all segments of the visceral peritoneum
(Figure 4). In addition,
disseminated focal areas of intense capillary proliferation that lacked
uniform spacing were observed, generally along the borders of the fat axes
that surround the larger mesenteric vessels
(Figure 5). These alterations
were absent in diabetic rats that were treated with anti-VEGF Ab, and the
peritoneal membranes of these rats were similar to those of control rats
(Figures 4 and
5).
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Quantification of the vascular density with image analysis software revealed an increased vessel/area ratio in the untreated diabetic and control Ab-treated diabetic rats but not in the anti-VEGF Ab-treated diabetic rats (Figure 6). The increase in vascular density was accounted for primarily by capillary proliferation: the relative contribution of capillaries to total vessel length was elevated substantially in untreated and control Ab-treated diabetic rats (Figure 6). VEGF blockade did not reduce total vessel length in control rats, although the percentage of capillaries was slightly lower than in untreated control rats (Figure 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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VEGF is known to induce angiogenesis through its potent stimulating effect on endothelial cell proliferation, migration, and tube formation (9,10). In the diabetic rats, evidence for neoangiogenesis was observed with different techniques. After 6 wk of diabetes, peritoneal vascular density was elevated substantially, with both an increased density of the mature vascular network and the presence of focal areas of extensive capillary budding. After 1 wk of diabetes, focal capillary proliferation was already present, but the density of the established vascular network was normal (De Vriese AS, unpublished observations), suggesting that the capillary budding precedes the development of vascular networks. Systematic quantification of the recordings made during intravital microscopy revealed that vascular density in the diabetic rats exceeded that observed in control rats and that the relative contribution of capillaries was higher. The functional consequences of the enlarged peritoneal vascular surface area were highlighted by an increased transport of small molecular weight solutes. Administration of anti-VEGF Ab dramatically inhibited the neoangiogenesis in the diabetic rats, with a commensurate effect on the transport of small solutes. These results thus implicate VEGF as a key mediator of hyperglycemia-induced neoangiogenesis.
VEGF also is a very potent enhancer of microvascular permeability. The exact mechanisms and structures involved in the permeability-enhancing effect of VEGF are still controversial. Three mechanisms have been implicated: (1) induction of endothelial fenestrations, (2) functional activation of vesicularvacuolar organelles in the cytoplasm of endothelial cells, and (3) stimulation of the formation of interendothelial gaps. It has been proposed that the increase in microvascular permeability to proteins induced by VEGF is an essential step in angiogenesis, allowing the extravasation of blood-borne proteins and the formation of a matrix to support the growth of the endothelial cells and formation of tubes (9). Leakage of macromolecules was studied at the level of the individual microvessel and found to be significantly increased in the diabetic rats, as described previously (29). It should be noted that the present approach does not allow discrimination of the relative contribution of microvascular hyperpermeability and microvascular hypertension to the development of macromolecular leakage. Blockade of VEGF reduced early macromolecular leakage to values not different from those in control rats, supporting an important role for VEGF. At the end of the observation period, FITC-albumin leakage was somewhat higher in anti-VEGF Abtreated diabetic rats than in control rats, suggesting the involvement of other mechanisms. Oxidative stress (30), advanced glycation end products/receptor for advanced glycation end products interaction (31), and increased activity of the polyol pathway (32) have been implicated as mediators of increased microvascular permeability in diabetes and may have played a contributing role in our model. Application of exogenous VEGF rapidly increases permeability in different tissues (12,13), revealing the presence of functional receptors on quiescent endothelial cells. It has been suggested, therefore, that VEGF is responsible for inducing and maintaining the baseline permeability of the normal microcirculation. The present results do not support a role for VEGF in normal permeability for macromolecules, inasmuch as VEGF blockade did not affect leakage of FITC-albumin in control rats.
Intravascular administration of VEGF induces endothelium-dependent vasodilation and results in a reduction of BP (12,33). VEGF is, however, primarily a paracrine mediator, and the significance of systemic VEGF levels is undefined. The presence of VEGF in serum samples is due mainly to VEGF release from platelets upon their activation during coagulation (34). True circulating VEGF levels are actually very low, which is not surprising in view of the extreme potency of the molecule and the dramatic consequences of its actions. We failed to demonstrate any effect of chronic VEGF antagonism on BP, supporting a limited hemodynamic role for endogenous free circulating VEGF.
The prevention of hyperglycemia-induced microvascular changes by VEGF blockade suggests an increased expression of, activity of, or sensitivity to this growth factor. An increased expression of VEGF has been demonstrated previously in the target organs for microvascular dysfunction in humans and experimental animals with diabetes (17,18,19,20,21). Local generation of VEGF was reported in the peritoneum of patients who were exposed to glucose-based peritoneal dialysate (22). The cause of the upregulation of VEGF by high glucose remains speculative, but multiple factors may be implicated. It has been suggested that high glucose concentrations, through an increased flux of glucose via the sorbitol pathway, may trigger hypoxia-like alterations in cellular redox status (23). Because tissue hypoxia is a major regulator of VEGF production, the hypoxia-like redox imbalance may inappropriately upregulate VEGF expression. In addition, glucose degradation products (35) and advanced glycation end products (36) are known to induce VEGF expression in vitro. The in vivo relevance of these pathways remains to be demonstrated.
An improved understanding of the role of VEGF in peritoneal microvascular alterations induced by high glucose concentrations is likely to be a source of therapeutic advance. Besides the implications for diabetic microvascular disease, the present results may be conducive to the understanding of the pathogenetic role of VEGF during long-term peritoneal dialysis. The functional and structural alterations observed in the peritoneal membrane of the diabetic rats were similar to those observed in chronic peritoneal dialysis patients with loss of ultrafiltration capacity. It should be noted that in diabetes, the peritoneal membrane is exposed to high glucose concentrations from the endothelial side, whereas during peritoneal dialysis the exposure occurs from the mesothelial side. The pathophysiologic consequences may be similar, however, because VEGF specifically targets endothelial cells, as only these cells carry functional VEGF receptors in vivo. Experimental diabetes therefore may be an appropriate in vivo model to study the effects of high dialysate exposure on peritoneal function and structure.
In conclusion, the present results indicate that VEGF represents a pivotal link between high glucose concentrations and microvascular hyperpermeability and neoangiogenesis in the peritoneal membrane. VEGF blockade, as currently tested in cancer patients (37), may constitute a novel, potentially useful therapeutic target to limit the microvascular complications induced by high glucose concentrations.
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), control rats + anti-VEGF Ab (n
= 11, [UNK]), and diabetic rats + control Ab (n = 12, 
) before
and 2, 4, and 6 wk after the start of treatment. Inhibitory activities are
expressed as percentage of inhibition by 1:50 diluted serum of the binding
between 125I-VEGF and fms-like tyrosine kinase in vitro.
Comparison among time points: *, P < 0.001
versus 0 wk. Comparison among groups: #, P < 0.001
versus diabetic + anti-VEGF Ab and control + anti-VEGF Ab; 
,
P < 0.001 versus diabetic + anti-VEGF Ab.
),
anti-VEGF Ab-treated diabetic (
), control Ab-treated diabetic ([UNK]),
and anti-VEGF Ab-treated control rats (
