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Am J Physiol Gastrointest Liver Physiol 293: G591-G598, 2007. First published June 21, 2007; doi:10.1152/ajpgi.00572.2006
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MUCOSAL BIOLOGY

Role of VEGF in small bowel adaptation after resection: the adaptive response is angiogenesis dependent

Jignesh K. Parvadia,1,2,3 Sundeep G. Keswani,1,2,3 Sachin Vaikunth,1,2,3 Arturo R. Maldonado,1,2,3 A. Marwan,1,2,3 Wolfgang Stehr,2 Christopher Erwin,2 Eva Uzvolgyi,1,2,3 Brad W. Warner,2 Seichii Yamano,4 Norton Taichman,5 and Timothy M. Crombleholme1,2,3

1Center for Molecular Fetal Therapy, 2Divisions of Pediatrics General and Thoracic Surgery, and 3Experimental Hematology, Children's Hospital Medical Center, Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, Ohio; 4Harvard School of Dental Medicine, Boston, Massachusetts; and 5University of Pennsylvania, School of Dental Medicine, Philadelphia, Pennsylvania

Submitted 14 December 2006 ; accepted in final form 16 June 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Previous work in our group has demonstrated that mouse salivary gland has the highest concentration of salivary-derived VEGF protein compared with other organs and is essential for normal palatal mucosal wound healing. We hypothesize that salivary VEGF plays an important role in maintaining the integrity of the gastrointestinal mucosa following small bowel resection (SBR). Thirty-five 8- to 10-wk-old C57BL/6 female mice were divided into seven treatment groups: 1) sham (transaction and anastomosis, n = 5); 2) SBR (n = 8); 3) sialoadenectomy and small bowel resection (SAL+SBR, n = 8); 4) sialoadenectomy and small bowel resection with EGF supplementation (SAL+SBR+EGF, n = 9); 5) sialoadenectomy and small bowel resection with VEGF supplementation (SAL+SBR+VEGF, n = 9); 6) sialoadenectomy and small bowel resection supplemented with EGF and VEGF (SAL+ SBR+VEGF+EGF, n = 6); 7) selective inhibition of VEGF in the submandibular gland by Ad-VEGF-Trap following small bowel resection (Ad-VEGF-Trap+SBR, n = 7). Adaptation was after 3 days by ileal villus height and crypt depth. The microvascular response was evaluated by CD31 immunostaining and for villus-vessel area ratio by FITC-labeled von Willebrand factor immunostaining. The adaptive response after SBR was significantly attenuated in the SAL group in terms of villus height (250.4 ± 8.816 vs. 310 ± 19.35, P = 0.01) and crypt depth (100.021 ± 4.025 vs. 120.541 ± 2.82, P = 0.01). This response was partially corrected by orogastric VEGF or EGF alone. The adaptive response was completely restored when both were administered together, suggesting that salivary VEGF and EGF both contribute to intestinal adaptation. VEGF increases the vascular density (6.4 ± 0.29 vs. 6.1 ± 0.29 vs. 5.96 ± 0.20) and villus-vessel area ratio (0.713 ± 0.01 vs. 0.73 ± 0.01) in the adapting bowel. Supplementation of both EGF and VEGF fully rescues adaptation, suggesting that the adaptive response may be dependent on VEGF-driven angiogenesis. These results support a previously unrecognized role for VEGF in the small bowel adaptive response.

sialoadenectomy; small bowel resection; VEGF; EGF

THE GASTROINTESTINAL MUCOSA is endowed with a remarkable capacity to heal rapidly despite continually being subjected to a variety of pathological insults. Although the defense mechanisms of these insults are not completely understood, saliva has been shown to play a significant role in maintaining oral mucosal homeostasis (25, 45). Numerous growth factors are secreted in high concentrations in the saliva. These include epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), nerve growth factor, transforming growth factor-{alpha} and -beta, acidic and basic fibroblast growth factors, and insulin-like growth factors (5). Together, these growth factors are thought to protect gastrointestinal mucosa by contributing multiple effects that restore tissues to health following insult or injury. Previous work in an animal model clearly demonstrates that the adaptive response of the gastrointestinal tract to massive small bowel resection (SBR) is critically dependent on salivary-derived EGF (17). The role of other salivary-derived growth factors in this adaptive response has, however, not been investigated.

Angiogenesis, the growth of new blood vessels from preexisting blood channels, is an absolute requirement for successful healing of damaged tissues. Proliferation of new vessels results in the expansion of the microvascular bed, enabling the influx of more leukocytes, nutrients, growth factors, oxygen, and other metabolites needed to initiate and sustain healing and repair (5, 29, 30). Adaptation following massive SBR may possibly be due to an endothelial response. This response could involve salivary-derived growth factors and salivary peptides that are either directly or indirectly angiogenic.

VEGF-A is a protein found in high concentration in healthy human and murine saliva. This protein is primarily derived from the parotid gland in humans, whereas in mice the submandibular glands are the principal source (25, 45). VEGF-A is a potent direct angiogenic peptide that may be pivotal in upregulating microvascular proliferation in oral and gastrointestinal mucosa. VEGF-A and angiogenesis are both known to be locally upregulated in dermal (11, 12, 15) and gastrointestinal wounds (11, 12, 15, 26, 32, 39, 40).

The loss of intestinal mucosal surface area is a relatively common clinical situation in both children and adults. It is frequently caused by mesenteric ischemia, trauma, inflammatory bowel disease, necrotizing enterocolitis, and volvulus and often is treated by SBR. Following massive SBR, the remnant intestine compensates for the loss of native bowel by increasing its absorptive surface area and functional capacity. Adaptation is characterized grossly by both increased length and caliber, morphologically by hyperplasia and hypertrophy of all intestinal layers, and functionally by augmented absorptive and digestive capacity (10). Although the exact mechanisms and/or mediators of this important response remain unclear, there is evidence that EGF and its intestinal receptor (EGF-R) are crucial components of adaptation (9). EGF is a heat-stable peptide that is a potent mitogen for several epithelial cell lines. It is known to play a role in the normal growth, development, and maturation of gastrointestinal tract (1, 11). EGF induces proliferation of skin, lung, and tracheal, corneal, and gastrointestinal epithelium (46). Additionally, it accelerates the tensile strength of surgical wounds and helps to maintain normal intestinal architecture (7). In animal models, administration of EGF after SBR has been shown to augment intestinal adaptation (9). Nevertheless, the specific site of action for endogenous salivary-derived EGF is not well understood. It is known that enterocytes express VEGF-2 (also known as Flk-1) receptors and that there are high levels of VEGF in breast milk (35). Submandibular gland excision results in a variety of gastrointestinal effects, including reduced DNA synthesis in oxyntic mucosa and pancreas (33). Interestingly, these effects are reversed following parenteral administration of aqueous extracts of the excised salivary glands (32). These findings suggest that salivary VEGF may have a direct effect on the gastrointestinal mucosal adaptive response. The effects of salivary gland excision and administration of VEGF on small bowel adaptation after sialoadenectomy (SAL) have not been reported.

Collective findings pertaining to the relationship between salivary-derived VEGF and small bowel adaptation led us to hypothesize that salivary VEGF plays an important role in maintaining the integrity of the gastrointestinal mucosa following SBR. To test this hypothesis, we performed a series of experiments. Our objectives were to determine 1) the effect of SAL on intestinal adaptation after SBR; 2) the effect of selective inhibition of VEGF by Ad-VEGF-Trap (VEGF inhibitor) on intestinal adaptation after SBR; 3) whether the effect of SAL and selective inhibition of VEGF could be reversed by administration of either VEGF or EGF alone or VEGF and EGF combined; and 4) the effect of administration of VEGF on angiogenesis, as demonstrated by submucosal and villus microvasculature density following SBR.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Fifty-two 8- to 10-wk-old C57BL/6 female mice (Jackson Laboratory, Bar Harbor, ME) were housed at 21°C on 12-h day-night cycles (6 AM to 6 PM). Before experimentation, the mice were allowed to acclimate to their environment over a 5-day period. A protocol for this study was approved by the Children's Hospital Research Foundation Institutional Animal Care and Use Committee.

Study design. To determine whether salivary-derived VEGF is important in adaptation following SBR, mice were divided into seven treatment groups. These groups were comprised of 1) sham (transaction and anastomosis, n = 5); 2) small bowel resection (SBR, n = 8); 3) sialoadenectomy and small bowel resection (SAL+SBR, n = 8); 4) sialoadenectomy and small bowel resection with EGF supplementation (SAL+SBR+EGF, n = 9); 5) sialoadenectomy and small bowel resection with VEGF supplementation (SAL+SBR+VEGF, n = 9); 6) sialoadenectomy and small bowel resection supplemented with EGF and VEGF (SAL+SBR+VEGF+EGF, n = 6); and 7) selective inhibition of VEGF in the submandibular gland by Ad-VEGF-Trap following small bowel resection (Ad-VEGF-Trap+SBR, n = 7).

Mice were harvested on the third postoperative day at the same time each day to avoid confounding factors. This time point was based on previous work demonstrating that the adaptive response to SBR is maximal and sustained by this time (18). At the time of euthanasia, 6 cm (~1 cm distal from the anastomosis) of the ileum was excised. The luminal content was gently expressed with cotton swabs. The proximal 1 cm of the section was fixed in 10% neutral buffered formalin and used for histology and immunohistochemistry; the remaining 5 cm of the section was utilized for the measurement of DNA content.

EGF. Purified human recombinant EGF (Sigma-Aldrich, St. Louis, MO) was administered in a dose of 50 µg/kg twice daily (at 0800 and 2000) via the orogastric route for 3 days as previously described (34).

VEGF. Oral VEGF (mBA-165 purified mouse VEGF protein, 50 µg, Santa Cruz Biotechnology, Santa Cruz, CA) was administered in a dose of 100 ng/ml. This was based on data obtained from the same strain of mouse used in previous studies related to VEGF and was chosen because it was within the range of 0.5–100 ng/ml as an estimate of physiological levels.

VEGF and EGF. A cocktail combining the above mentioned doses of EGF (50 µg·kg–1·day–1) and VEGF (100 ng/ml) was administered twice daily via the orogastric route.

Ad-VEGF-Trap. An adenoviral vector was used to deliver a novel protein called VEGF-Trap (generously provided by John Rudge, Regeneron, Tarrytown, NY) to selectively inhibit salivary-derived VEGF. The Ad-VEGF-Trap produces transgene, which consists of VEGF receptors Flt-1 and Flk-1 bound to the Fc portion of an IgG1. This inhibitor binds VEGF with high affinity and has a prolonged in vivo half-life. Ad-VEGF-Trap was administered through a generation E1 E3 deleted replication incompetent adenoviral construct under the control of cytomegalo virus (CMV) promoter at 1 x 108 particle-forming units in 50 µl of vector via retrograde injection in each salivary duct simultaneously as previously described (45). SBR was performed within 48 h of Ad-VEGF-Trap administration. This time frame was chosen on the basis of previous work in our laboratory demonstrating that expression of the transgene in salivary gland is confined to a 48-h period.

SAL. SAL was performed as previously described (17). Briefly, through a midline cervical incision, the bilateral paired submandibular salivary glands were freed of surrounding tissue, each vascular pedicle was cauterized with heat cautery, and the glands were excised. Skin was closed with skin adhesive. Mice were resuscitated with subcutaneous injection of normal saline solution (3 ml) and allowed to recover in a warmed incubator (33°C). Mice were fed with a liquid diet and underwent SBR 48 h after SAL.

SBR. SBR was performed as previously described (18). Briefly, mice were anesthetized with inhaled isoflurane anesthesia; the abdomen was shaved, prepped, and draped; and a midline abdominal incision was performed. An operating microscope was used for magnification (original magnification x10 to x15). In mice undergoing sham operation, the small bowel was transected and reanastomosis was performed 12 cm proximal to the ileocecal valve. In mice undergoing SBR, 12.5 cm of proximal intestine was resected and anastomosis was accomplished (50% resection). After abdominal closure, the mice were resuscitated with an intraperitoneal injection of 0.9% saline solution (3 ml) and allowed to recover in a warmed incubator (33°C). After 2 h, they were returned to their regular facilities individually caged. Clear water was provided ad libitum for the first 24 h. Mice from each group were then pair-fed with a liquid diet (Micro-stabilized rodent liquid diet LD 101/101; Purina Mills, Richmond, IN).

Histology. Formalin-fixed specimens of ileum were embedded in paraffin and oriented to provide cut sections parallel with the longitudinal axis of the bowel. Five-micrometer sections were cut on a microtome (Leica RM 2135) and collected on super frost plus slides. Sections were deparaffinized, serially rehydrated, and stained with hematoxylin and eosin. By use of the hematoxylin and eosin-stained sections villus height and crypt depth were measured with a computer-assisted integrated computer program (Nikon-80i-Eclipse, Metamorph, Fryer, Warrensville Heights, OH) as previously described (18). At least 12–15 villi and crypts were counted per sample. Villi were selected only if the central lymphatic channel was completely visualized and crypts were selected only if the crypt-villus junction on both sides of the crypt was demonstrated in the section. All histology was performed with the investigator blinded as to the source of ileal tissue.

Immunohistochemical staining. Rehydrated paraffin sections (5 µm) were washed in phosphate-buffered saline (PBS) and endogenous peroxidase activity was blocked in 3% H2O2 in methanol at room temperature (RT) for 10 min. The slides were immersed in 0.1% trypsin (GIBCO 1:250, Invitrogen) to reactivate hidden or masked epitopes and were placed in a 37°C water bath. Samples were washed in distilled water followed by PBS and then blocked with nonimmune rabbit serum. This was followed by incubation with a monoclonal rat anti-mouse CD31 (1:20, Pharmingen, San Diego, CA) at 4°C overnight. Slides were rinsed in 0.1 M PBS-0.1% Triton X-100 and incubated with biotinylated rabbit anti-rat (1:200; Vector Laboratories, Burlingame, CA) adsorbed at RT for 30 min. Sections were washed several times in PBS and treated with avidin-biotin complex (Elite, Vector Laboratories) for 30 min at RT. The slides were rinsed in PBS, developed with chromogen 3,3'-diaminobenzidine (Vector Laboratories) for permanent visualization, washed in distilled water, and counterstained with hematoxylin.

The stained microvessels in mucosal and submucosal area of small bowel were counted manually under light microscopy at x40 magnification using 10 randomly chosen fields from each slide, as previously described (18).

Immunofluorescence analyses (38) were performed using a primary antibody to von Willebrand factor (vWF). Briefly, deparaffinized specimens were rehydrated in a series of graded ethanol. Sections were rinsed in PBS. This was followed by blocking of endogenous peroxide activity in 3% H2O2 in methanol at RT for 10 min. Antigen retrieval was performed by immersing slides in 0.1% trypsin in a 37°C water bath. Sections were then blocked in 1x goat serum dilution buffer overnight at 4°C. On the following day, polyclonal rabbit anti-human vWF (1:100, Chemi-Con, Temecula, CA) antibody was applied and sections were incubated at RT for 2 h. The slides were washed in wash buffer and incubated in goat anti-rabbit IgG Alexa Fluor 488 (1:200, Molecular Probes, Eugene, OR) at RT for 2 h. Slides were rinsed in wash buffer followed by incubation in 5 M NaPO4 buffer. Sections were coverslipped in Vectashield mounting medium (Vector Laboratories) and sealed with nail polish. The slides were kept in dark at 4°C until used. Under fluorescence microscopy at x20 magnification and 10 high-power fields (Nikon-80i-Eclipse, Metamorph, Fryer) the ratio between the area of vWF-stained vessel and villus was generated (GoGoGoGoGoGoFig. 7).


Figure 1
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  		Fig. 1. Hematoxylin and eosin (H&E)-stained section of mouse ileum (original magnification x20) 3 days postoperatively. A: 50% proximal small bowel resection (SBR). Complete adaptive response. B: bilateral submandibular gland excision [sialoadenectomy (SAL)] and SBR. Attenuated adaptive response. C: 50% proximal SBR and bilateral SAL followed by human recombinant VEGF replacement (100 ng/ml twice daily orogastric route) after SAL+SBR. D: combination of both VEGF and EGF replacement (50 µg·kg–1·day–1) after SAL+ SBR. On regular H&E, the SBR group has significantly increased villus height compared with sham. SAL group has significant decreases in villus height compared with SBR group. VEGF supplementation group has significant increase in the villus height compared with SAL group.

 

Figure 2
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  		Fig. 2. A: effect of SAL and exogenous VEGF and EGF on villus height after resection. Mice undergoing SBR alone demonstrated an adaptive response characterized by a significant increase in villus height compared with sham-operated control mice. In contrast to the adaptive response exhibited by mice undergoing SBR alone, mice undergoing SAL and SBR in the adaptive responses are significantly blunted by reduced villus height. Exogenous supplementation with either VEGF-A or EGF following SAL and SBR partially corrected the villus height compared with SBR without SAL. The combination of exogenous VEGF and EGF following SAL and SBR fully restored the adaptive response to SBR as indicated by significantly increased villus height (*P < 0.05 for comparisons of mice undergoing SBR vs. SAL+SBR, SBR vs. SAL+SBR+VEGF and SAL+SBR+EGF and SAL+SBR+VEGF+EGF). B: effect of SAL and exogenous VEGF and EGF on crypt depth after resection. Mice undergoing SBR also showed a significant increase in crypt depth compared with sham-operated control mice. In contrast to the adaptive response exhibited by mice undergoing SBR alone, mice undergoing SAL and SBR the adaptive response were significantly blunted as indicated by reduced crypt depth. Exogenous supplementation with either VEGF-A or EGF following SAL and SBR partially corrected the crypt depth compared with SBR without SAL. The combination of exogenous VEGF and EGF following SAL and SBR fully restored the adaptive response to SBR as indicated by significantly increased crypt depth (*P < 0.05 for comparisons of mice undergoing SBR vs. SAL+SBR, SAL+SBR vs. SAL+SBR+VEGF and SAL+SBR+EGF and SAL+SBR+VEGF+EGF). C: effect of SAL on DNA content. DNA content was also significantly increased in mice undergoing SBR compared with sham-operated control mice. In contrast to the adaptive response exhibited by mice undergoing SBR alone, in mice undergoing SAL and SBR the adaptive responses are significantly blunted as indicated by decreased DNA content (*P < 0.05 for comparisons of mice undergoing SBR vs. sham and SBR vs. SAL+SBR).

 

Figure 3
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  		Fig. 3. A: effect of Ad-VEGF-Trap-treated salivary glands on villus height. Retrograde injection of Ad-VEGF-Trap with preservation of the salivary glands resulted in a significant reduction in villus height (*P < 0.05 for comparisons of mice undergoing SBR vs. SAL+SBR). Intraperitoneal VEGF supplementation completely corrects the adaptive response by significant increase in villus height after Ad-VEGF-Trap injection and SBR (*P < 0.05 for comparisons of mice undergoing SAL+SBR vs. Ad-VEGF-Trap+SBR+VEGF). B: effect of Ad-VEGF-Trap-treated salivary glands on crypt depth. Retrograde injection of Ad-VEGF-Trap with preservation of the salivary glands results in a significant reduction in crypt depth (*P < 0.05 for comparisons of mice undergoing SBR vs. SAL+SBR). Intraperitoneal VEGF supplementation completely corrects the adaptive response with significant increase in villus height after Ad-VEGF-Trap injection and SBR (*P < 0.05 for comparisons of mice undergoing SAL+SBR vs. Ad-VEGF-Trap+SBR+ VEGF).

 

Figure 4
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  		Fig. 4. CD31 (PECAM-1)-immunostained section of mouse ileum (magnification x20). A: 50% proximal SBR. Complete adaptive response. B: bilateral SAL and SBR. Attenuated adaptive response. C: 50% proximal SBR and bilateral SAL followed by human recombinant VEGF replacement (100 ng/ml twice daily orogastric route) after SAL+SBR. D: combination of both VEGF and EGF replacement (50 µg·kg–1·day–1) after SAL+ SBR. Microvessels revealed by anti-CD31 immunostaining of mouse ileum from the VEGF-supplemented group showing robust capillary in growth. Mucosal and submucosal microvessel density was counted per high-power fields at x40 magnification. Mice undergoing SAL and SBR that were supplemented with VEGF have submucosal capillary density restored to the value observed in the sham or SBR mice. Supplementing mice with both EGF and VEGF also increased the submucosal capillary density to high values comparable to those decreased in sham-operated mice and mice undergoing SBR alone.

 

Figure 5
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  		Fig. 5. Effect of exogenous orogastric VEGF on submucosal capillary density. Mice undergoing SAL and SBR have a significant reduction in submucosal capillary density compared with mice undergoing SBR alone or sham operation. The reduction in submucosal capillary density observed in SAL mice undergoing SBR is partially restored by orogastrically supplemented EGF but still significantly lower capillary density compared with SBR controls. Mice undergoing SAL and SBR that were orogastrically supplemented with VEGF have submucosal capillary density restored to the value observed in the sham or SBR mice. Supplementing mice with both EGF and VEGF also increased the submucosal capillary density comparable to those in sham-operated mice and mice undergoing SBR alone. There was increase in capillary density in mice treated with VEGF and EGF opposed to mice supplemented with EGF alone (*P < 0.05 for comparisons of mice undergoing SBR vs. SAL+SBR, SAL+SBR vs. SAL+SBR+VEGF and SAL+SBR+VEGF+EGF).

 

Figure 6
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  		Fig. 6. FITC-von Willebrand factor (vWF)-immunostained section of mouse ileum (magnification x20). A: 50% proximal SBR. Complete adaptive response. B: bilateral SAL and SBR. Attenuated adaptive response. C: 50% proximal SBR and bilateral SAL followed by human recombinant VEGF replacement after SAL+SBR. D: combination of both VEGF and EGF replacement after SAL+SBR. Under fluorescence microscopy at x20 magnification and 10 high-power fields the ratio between area of fluorescent and vWF-stained vessel vs. villi was generated. This image shows fluorescent-stained villus and center of each villus stained with vWF, suggesting the vessel of the corresponding villus.

 

Figure 7
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  		Fig. 7. Effect of SAL and exogenous orogastric VEGF supplementation on vessel-to-villus area ratio. To assess the vascular density of villi during bowel adaptation following SBR, vessel-to-villus area ratio was made. SAL results in a statistically significant reduction in vessel-villus area ratio in mice undergoing SBR compared with mice undergoing SBR alone. VEGF supplementation following SAL and SBR corrected vessel-villus area ratio to control levels, as did supplementing with VEGF and EGF, but supplementing with EGF alone had no effect on vessel-villus area ratio. The correction of submucosal capillary density and vessel-to-villus area ratio by supplemented VEGF suggests that the effect of VEGF on bowel adaptation following SBR is mediated through its angiogenic effects (*P < 0.05 for comparisons of undergoing SBR vs. SAL +SBR, SAL+SBR vs. SAL+SBR+VEGF and SAL+SBR+VEGF+EGF).

 
VEGF ELISA. After induction of anesthesia preceding death, blood was obtained via cardiac puncture and placed on ice. Blood samples were centrifuged at 10,000 g for 10 min and serum collected for the ELISA analysis of VEGF-A levels (Quantikine mouse VEGF, R&D Systems, Minneapolis, MN). Concentrations of VEGF in serum were determined with use of standard curves derived from the known concentration of mouse VEGF.

Statistics. Results are presented as means ± SE unless otherwise stated. Mean values between all groups were analyzed by one-way ANOVA. Individual group differences were analyzed by Tukey's post hoc test. A P value of <0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Thirty-five of 52 mice were healthy at the time of harvest and thus included in the analysis. Survival rates were as follows: sham-operated animals 5 of 5 (100%); SBR 5 of 8 (62.5%); SAL+SBR 5 of 8 (62.5%); SAL+SBR+VEGF (5 of 9) 55.5%; SAL+SBR+EGF (5 of 9) 55.5%; SAL+SBR+VEGF+EGF (5 of 6) 83.3%, Ad-VEGF-Trap+SBR (5 of 7) 71.42%.

SAL attenuated adaptation following SBR. Mice that underwent SBR alone demonstrated an adaptive response. This response was characterized by a significant increase in villus height compared with sham-operated controls (310 ± 19.3 vs. 230.4 ± 4.7, P = 0.001, Fig. 1A). These mice also showed a significant increase in crypt depth compared with sham-operated controls (120.541 ± 2.82 vs. 80.83 ± 1.05, P = 0.001). Additionally, DNA content of SBR-alone mice showed significant increase compared with sham-operated controls (166.83 ± 48.34 vs. 519.19 ± 72.85, P = 0.002). In contrast to the adaptive response exhibited by mice that underwent SBR alone, animals that underwent SAL+SBR showed a significantly blunted adaptive response (Fig. 1B) compared with the SBR group in villus height (250.4 ± 8.81 vs. 310 ± 19.35, P = 0.01, Fig. 2A), crypt depth (100.021 ± 4.02 vs. 120.54 ± 2.82, P = 0.01, Fig. 2B), and DNA content (290.78 ± 35.38 vs. 519.19 ± 72.85, P = 0.001, Fig. 2C).

Selective VEGF inhibition attenuated adaptive response following SBR. To examine the contribution of salivary-derived VEGF to adaptation, we selectively inhibited VEGF. Retrograde injection of Ad-VEGF-Trap with preservation of the salivary glands resulted in a significant reduction in both villus height (202.3 ± 28.1 vs. 310 ± 19.35, P = 0.002, Fig. 3A) and crypt depth (96.11 ± 1.73 vs. 120.54 ± 2.82, P = 0.001, Fig. 3B) compared with the SBR group. This was consistent with the blunted adaptive response seen with SAL.

Administration of exogenous EGF and VEGF restored the adaptive response following SAL+SBR. Exogenous supplementation of either VEGF-A or EGF following SAL+SBR only partially restored the adaptive response as indicated by villus height [VEGF 266 ± 6.7 vs. 310 ± 19.35, P = 0.004 (Fig. 1C); EGF 259.2 ± 2.69 vs. 310 ± 19.35, P = 0.003 (Fig. 2A)] compared with SBR alone. Exogenous supplementation of VEGF or EGF alone following SAL+SBR partially restored the adaptive response as indicated by crypt depth (VEGF 114.9 ± 3.9 vs. 120.54 ± 2.821, P = 0.004, EGF 109.03 ± 1.35 vs. 120.54 ± 2.82, P = 0.003) compared with SBR alone (Fig. 2B).

The combination of exogenous VEGF and EGF following SAL+SBR fully restored the adaptive response to SBR, as indicated by significantly increased villus height (271.8 ± 16.46 vs. 310 ± 19.35, P = 0.84, Figs. 1D and 2A) and crypt depth (118.18 ± 6.6 vs. 120.54 ± 2.82, P = 0.62, Fig. 2B).

Exogenous VEGF supplementation restored capillary density in SAL mice following SBR. Mice that underwent SAL+SBR demonstrated a significant reduction in submucosal capillary density compared (Figs. 4B and 5) with those that underwent SBR alone (3.68 ± 0.35 vs. 5.96 ± 0.20, P = 0.03, Fig. 4A) or sham operation (3.68 ± 0.35 vs. 6.1 ± 0.29, P = 0.03). The reduction in submucosal capillary density observed in SAL+SBR was partially restored by supplemented EGF (4.96 ± 0.49 vs. 3.68 ± 0.35), although the capillary density was still significantly lower than that observed in SBR alone (4.96 ± 0.49 vs. 5.96 ± 0.20, P = 0.01). Mice that underwent SAL+SBR and that received supplementation with VEGF had submucosal capillary density restored (Fig. 4C) to the value observed in the sham-operated control and SBR mice [6.4 ± 0.29 vs. 6.1 ± 0.29 vs. 5.96 ± 0.20, P = not significant (ns)]. Supplementation with both EGF and VEGF also augmented submucosal capillary density to levels comparable to those observed in sham-operated mice and mice that underwent SBR alone. Also, there was an increase in capillary density in mice treated with VEGF and EGF compared with mice supplemented with EGF alone (Fig. 4D).

Supplementation of VEGF corrected SAL-induced reduction in vessel-to-villus area ratio. To assess the vascular density of villi during bowel adaptation following SBR, vessel-villus area ratio was measured as previously described (38). SAL resulted in a statistically significant reduction in vessel-villus area ratio in mice following SBR (Fig. 6B) compared with mice that underwent SBR alone (Fig. 6A) (0.66 ± 0.01 vs. 0.73 ± 0.01, P = 0.0001). VEGF supplementation following SAL+SBR corrected (Fig. 6C) the vessel-villus area ratio to the level exhibited in control (0.713 ± 0.01 vs. 0.73 ± 0.01 in SBR vs. 0.702 ± 0.01 in sham;, P = ns).

Similarly, supplementation with both VEGF and EGF (Fig. 6D) corrected vessel-villus area ratios to the level exhibited in control (0.724 ± 0.001 vs. 0.73 ± 0.01 in SBR vs. 0.702 ± 0.01 in sham, P = ns); however, supplementation with EGF alone had no effect on vessel-villus area ratio. The correction of submucosal capillary density and vessel-villus area ratio by supplementation of VEGF suggests that the effect of VEGF on bowel adaptation following SBR is mediated through its proangiogenic effects (Fig. 7).

VEGF level in serum. Serum VEGF levels were not affected by SAL+SBR. Orogastric supplementation of either VEGF or EGF alone or combined had no significant effect on serum VEGF levels.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our results suggest a novel, unrecognized role played by VEGF in the adaptive response of the small intestine. We report that salivary-derived VEGF is essential for adaptive response following massive SBR. Complete restoration of the adaptive response is observed only with combined administration of VEGF and EGF. These results may potentially have a significant impact on patient undergoing massive SBR; administration of both VEGF and EGF may increase rapidity of their adaptive response, ultimately improving their overall functional gut recovery.

However, to date most of the published literature on small bowel adaptation has focused on the role of EGF. Previous work by Warner et al. (9) has established that EGF is necessary for small bowel adaptation after massive SBR. Removal of the salivary gland, the major source of both VEGF and EGF in mice, leads to a decrease in the adaptive response with respect to villus height, crypt depth, and DNA content. Selective inhibition of VEGF shows a decrease in the adaptive response similar to decreases seen in mice that have undergone SAL+SBR. Simultaneous administration of VEGF and EGF completely restores the adaptive response with respect to villus height, crypt depth, and DNA content.

The specific importance of salivary-derived VEGF, as opposed to other salivary-derived growth factors in the adaptive response to massive SBR, is supported by the effects of selective inhibition of VEGF. This is further supported by results of orogastric administration of exogenous VEGF, which corrects the adaptive response after either SAL or selective inhibition of VEGF. This finding shows that VEGF administration leads to increased adaptation parameters (villus height, crypt depth) but does not, however, completely restore the adaptive response to SBR baseline. This suggests that VEGF may play both a direct and an indirect role in allowing adaptive response. Positive effects of VEGF supplementation may be mediated either via luminal expression of VEGF-R in the gastrointestinal tract, via the portal venous system, or via gut lymphatic system, but not, however, via systemic circulation. These results demonstrate that VEGF supplementation following SAL+SBR leads to a significant increase in the submucosal vascular density as well as the vessel-villus area ratio compared with EGF supplementation following SAL+SBR.

Whereas VEGF is known to be a key player in neovascularization, the function of salivary-derived VEGF remains unclear. Previous studies (11, 12, 15, 32) have demonstrated that salivary-derived VEGF significantly contribute to the mechanisms that defend and repair the mucosa of the oral cavity as well as other parts of the alimentary tract. Given the importance of VEGF in oral mucosal integrity, wound healing and neovascularization are potentially important in the adaptive response to massive SBR. VEGF appears to have a distinct but equally important role in the intestinal adaptive response to SBR through its angiogenic effects. These effects augment the vascular development of the submucosa. Unpublished data from our laboratory further support the role of salivary-derived VEGF in gastrointestinal angiogenesis. We have recently shown that SAL significantly impairs the compensatory liver growth and neovascularization following partial hepatectomy. This is rescued by orogastric or intraperitoneal supplementation of VEGF.

Numerous studies (17, 18) have documented support for EGF as necessary and sufficient to correct adaptation to baseline levels following SAL. These studies have, however, used a 7-day time point. In contrast we used a 3-day time point. This time point was chosen on previous work in SBR animal model on the basis of completion of the adaptive response by 3-day period (18). Differences in findings between previous studies with EGF supplementation and present studies are likely to reflect the differences in time point selected. The 7-day time point may allow sufficient time for EGF alone to stimulate the adaptive response. The combined supplementation of VEGF and EGF supplementation may increase the velocity of adaptive response by stimulating the submucosal neovasculature to keep pace with the demands of the overlying mucosa undergoing adaptation.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research study was supported by grants from the National Institutes of Health (DK-59242, DK-0740055, DKO-72446, Juvenile Diabetes Research Foundation and American Diabetes Association, T. M. Crombleholme).


   ACKNOWLEDGMENTS
 
We thank Datis Alaee, Barbara Kalinowska, and Maria Ripberger for supplying the material and technical help for vector, histology, and immunohistochemistry. The generous gift of Ad-VEGF trap construct from John Rudge, PhD, Regeneron is gratefully acknowledged.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. M. Crombleholme, Center for Molecular and Fetal Therapy, Division of Pediatric General, Thoracic, and Fetal Surgery, Cincinnati Children's Hospital Medical Center, 3333 Burnet Ave. MLC 2023, Cincinnati, OH 45229-3039 (e-mail: timothy.crombleholme{at}cchmc.org)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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