In vivo non-viral gene delivery of human vascular endothelial growth factor improves revascularisation and restoration of euglycaemia after human islet transplantation into mouse liver
- First Online:
- 507 Downloads
Delivery of the gene for human vascular endothelial growth factor (VEGF, also known as VEGFA) to both the transplanted islets and the surrounding tissue may promote islet revascularisation and survival. We previously showed the effective delivery of VEGF gene to rat myocardium by an ultrasound-mediated gene-transfer method named ultrasound-targeted microbubble destruction (UTMD). Here we examined the effect of non-viral VEGF delivery using UTMD on transplanted islets in vivo.
A marginal number of human islets were transplanted into livers of mice which were a model for diabetes. Then, non-viral plasmid vectors encoding VEGF (VEGF group, n = 11) or the gene for green fluorescent protein (GFP) (GFP group, n = 7) were introduced into the host liver by UTMD. Transplantation without gene delivery was performed as a control (no-UTMD group, n = 8). Blood glucose, serum human insulin, C-peptide levels and the revascularisation in graft islets were evaluated.
Restoration of euglycaemia occurred in 13% in the no-UTMD group and 14% in the GFP group, whereas 73% mice in the VEGF group became euglycaemic at day 30 (p < 0.05 in no-UTMD vs VEGF). Serum human insulin and C-peptide were significantly higher in the VEGF group at day 32 (insulin: no-UTMD, 17 ± 8; GFP, 37 ± 17; VEGF, 109 ± 26 pmol/l, respectively, p < 0.05; C-peptide: no-UTMD, 68 ± 38; GFP, 115 ± 58; VEGF, 791 ± 230 pmol/l, respectively, p < 0.05). Vessel density in graft islets was significantly higher in the VEGF group (no-UTMD, 169 ± 36; GFP, 227 ± 39; VEGF, 649 ± 51 counts/mm2, respectively, p < 0.05).
Delivery of VEGF gene to host liver using UTMD promoted islet revascularisation after islet transplantation and improved the restoration of euglycaemia.
KeywordsGene therapy Human vascular endothelial growth factor Islet transplantation Non-viral gene delivery Revascularisation
Green fluorescent protein
Human vascular endothelial growth factor
Intraperitoneal glucose tolerance test
Ultrasound-targeted microbubble destruction
Islet transplantation is a promising treatment for type 1 diabetes; however, efficacy of transplantation needs to be improved, because currently multiple transplantation is required to achieve insulin-free status [1, 2]. It was shown that gene delivery to islets can improve the function and survival of islets [3, 4, 5, 6], but all previous studies used viral vectors. A viral vector has high efficacy for delivering genes, but adverse events have been related to enhancer-mediated mutagenesis of genomic DNA  or immunological responses to viral proteins .
In contrast, delivery of naked plasmid DNA (pDNA) does not transport toxic or immunogenic viral protein or polymer particles in vivo. However, plasmid vectors have been limited by low transfection efficiency. To obtain high gene expression with pDNAs, we have established a novel ultrasound-mediated gene-transfer method known as ultrasound-targeted microbubble destruction (UTMD). Microbubbles, which consist of a lipid shell encapsulating perfluorocarbon gas, are injected into the circulation. The pDNAs are incorporated into the lipid shell. Under ultrasound exposure, the microbubbles burst and create transient pores in the membranes of surrounding cells, and pDNAs are inserted into the cells. UTMD has many desired characteristics for gene therapy including low toxicity, low immunogenicity, potential for repeated application, organ specificity and broad applicability to acoustically accessible organs. We previously demonstrated that delivery of the gene (VEGF) for human vascular endothelial growth factor (hVEGF) to rat myocardium by UTMD resulted in significant increases in myocardial capillary and arteriolar density . Moreover, we reported that our UTMD technique enabled us to effectively deliver pDNAs to rat pancreatic beta cells in vivo [10, 11]. Lack of intra-islet microvasculature is one of the most important factors in loss of graft islets. Therefore, we hypothesise that early loss of islet grafts could be attenuated by delivery of the VEGF gene to promote revascularisation.
In this study, we used a transplantation model in which human islets were transplanted into mouse liver via the portal vein. This model is similar to the clinical setting. VEGF gene was delivered to the host liver by UTMD to examine whether it could facilitate revascularisation and improve the survival and function of the transplanted islets.
Seven donor pancreases were procured from deceased multiorgan donors after obtaining consent for research through the local Organ Procurement Organizations (Southwest Transplant Alliance, Dallas, TX, USA; LifeGift, Fort Worth, TX, USA). Baylor Regional Transplant Institute Surgeons using the standardised surgical technique performed pancreas procurement . Pancreases were preserved using the ductal injection method with ET-KYOTO solution (Otsuka Pharmaceutical Factory, Naruto, Japan) followed by a two-layer method until the islet isolation procedure . Islet isolation was performed using the modified Ricordi method . The donor and isolation variables were as follows: donor age 35.1 ± 5.6 years; two men and five women; BMI 28.0 ± 1.5 kg/m2; cold ischaemic time 171 ± 21 min; purity 77 ± 5%; viability 97 ± 1%; stimulation index 10.4 ± 3.4. The isolated islets were preserved in CMRL-supplemented culture medium (Cellgro, Manassas, VA, USA) containing 10% (vol./vol.) FBS (Atlanta Biologicals, Lawrenceville, GA, USA) at 4°C overnight. A total of 500 islets of similar size (approximately 200 μm) were hand-picked for transplantation. Our preliminary experiment showed 10–20% of mice became euglycaemic after transplantation of 500 islets. Therefore, we determined the number as a marginal.
Plasmids expressing the VEGF165 (also known as NRP2) gene under an enhanced CMV promoter (pCI-hVEGF) were made as follows: cDNA of VEGF165 was obtained as previously described . Briefly, a full-length cDNA of VEGF165 obtained from a healthy volunteer’s blood was PCR amplified by using the following PCR primers that contain a restriction site (the restriction sites are underlined): primer 1 (XhoI) 5′-TTCCTCGAGAATGAACTTTCTGCTGCTGTCTTG-3′; primer 2 (SmaI) 5′-AAACCCGGGTCACCGCCTCGGCTTGTCA-3′. The DNA was digested with XhoI and SmaI and then ligated into the corresponding sites of pCI mammalian expression vector (Promega, Madison, WI, USA). The plasmids were sequenced to confirm that no artefactual mutations were present. Plasmids encoding green fluorescent protein gene (GFP) under the enhanced CMV promoter (pCS2-GFP) were used as a control.
Manufacture of plasmid-containing lipid-stabilised microbubbles
Lipid-stabilised microbubbles were prepared as previously described in our laboratory . Briefly, a total of 200 μl DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine; Sigma, St Louis, MO, USA, 2.5 mg/ml) and DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine; Sigma, 0.5 mg/ml) solution, 5 μl 10% (wt/vol.) human albumin and 50 μl pure glycerol were added to 1.5 ml vials containing 2 mg plasmids dissolved in 50 μl Lipofectamine2000 (Invitrogen, Carlsbad, CA, USA) and mixed well and incubated at room temperature for 10 min, the remaining headspace was filled with the perfluoropropane gas (Air Products, Allentown, PA, USA) and then mechanically shaken for 30 s by a dental amalgamator (Vialmix; Bristol-Myers Squibb Medical Imaging, N. Billerica, MA, USA). The mean diameter and concentration of the microbubbles were measured by a particle counter (Beckman Coulter Multisizer III; Fullerton, CA, USA). Ten microlitres of microbubble solution, which consisted of approximately 50 μg of the plasmids, was diluted with 0.3 ml PBS just before the injection and injected into each mouse.
Animal protocol and UTMD
Hand-picked human islets (n = 500) were transplanted into the mouse liver through the ileo-caecal vein for each mouse. Then each mouse was randomly assigned to one of three groups. Microbubbles with plasmids were infused immediately after islet transplantation through the ileo-caecal vein in the GFP and VEGF groups for 60–90 s via a pump (Genie; Kent Scientific, Torrington, CT, USA).
During the infusion, ultrasound was directed to the liver using a commercially available ultrasound transducer (S3, Sonos 5500; Philips Ultrasound, Bothell, WA, USA). Ultrasound was then applied in ultraharmonic mode (transmit 1.3 MHz/receive 3.6 MHz) at a mechanical index of 1.4. Four bursts of ultrasound were triggered to every fourth end-systole by an electrocardiogram using a delay of 85 ms after the peak of the R wave. These settings have been shown to be optimal for plasmid delivery by UTMD using this instrument [9, 10, 11]. Bubble destruction was visually apparent in all mice (Fig. 1c, d). After infusion, the animals were allowed to recover. To study the efficacy of UTMD, the technique was performed for normal nude mice as described above without islet transplantation (n = 3). They were killed at 1–28 days after treatment for measurement of serum hVEGF levels with an ELISA kit (ALPCO Diagnostics, Salem, NH, USA). At day 3, the liver, pancreas, spleen, kidneys, stomach, lungs and heart were harvested for the assessment of gene expression by RT-PCR.
To evaluate the extent and area of hVEGF production in the transplanted islets, we transplanted islets into normal nude mice via the portal vein and then consecutively performed UTMD with VEGF plasmids. Three days after the treatment, the liver was harvested and examined by immunohistochemistry.
Effect of UTMD on liver
To evaluate liver toxicity and unwanted effects of hVEGF such as excessive liver angiogenesis and uncontrolled liver growth, UTMD with VEGF plasmid via the portal vein was performed as described above. The blood was collected at different time points after UTMD. Samples were analysed for the presence of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) by enzyme assay kits (Thermo Scientific, Waltham, MA, USA). Histological examination was performed after haematoxylin and eosin staining of formalin-fixed mouse liver sections isolated at 2, 7 and 30 days after UTMD.
Assessment of treated mice
Non-fasting glucose levels of each mouse were measured with blood glucose test strips (Precision Xtra; Abbott Diabetes Care, Alameda, CA, USA) up to 30 days after transplantation. When two consecutive blood glucose level measurements were <11.1 mmol/l, restoration of euglycaemia was considered to be present. Recurrence of diabetes was defined as return of the blood glucose to >11.1 mmol/l. At day 31, i.p. glucose tolerance testing (IPGTT) was performed. After overnight fasting, glucose (2 g/kg) was injected i.p. The blood glucose levels were measured for 120 min after injection. Animals were killed at day 32. The harvested liver was weighed and examined by immunohistochemistry. The blood was collected for measuring human insulin and human C-peptide levels with ELISA kits (ALPCO).
The tissue samples were frozen in liquid nitrogen and stored at −80°C. Total RNA was prepared from TRIzol (Invitrogen) according to the manufacturer’s instructions and was reverse transcribed using a SuperScript III First-Strand Synthesis System (Invitrogen). PCR was performed in 20 μl volume containing 1 μl cDNA, 10 μl HotStarTaq Master Mix (Qiagen, Valencia, CA, USA) and 10 pmol of each primer: GAPDH for human and Gapdh for mouse: 5′-CCCTTCATTGACCTCAACTACATG-3′(sense); 5′-TTCCATTGATGACAAGCTTCCC-3′(antisense); VEGF: 5′-AAGGAGGAGGGCAGAATCAT-3′(sense); 5′-ATCTGCATGGTGATGTTGGA-3′(antisense); mouse Vegf: 5′-ACGACAGAAGGAGAGCAGAAGT-3′(sense); 5′-CATGGTGATGTTGCTCTCTGAC-3′. The PCR conditions included denaturation at 95°C for 5 min, followed by 25–35 cycles of amplification by sequential denaturation at 94°C for 30 s and primer annealing as well as strand extension for 1 min. The RT-PCR products were then analysed on 2% (wt/vol.) agarose gels.
The collected tissues were fixed in 4% (wt/vol.) paraformaldehyde at 4°C overnight and equilibrated in 30% (wt/vol.) sucrose at 4°C overnight for cryoprotection. The tissues were cryopreserved with Tissue Tek optimal cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA, USA) at −80°C. Sections were cut at 10 μm increments and placed onto positively charged microscope slides. Sections were permeabilised with 0.1% (vol./vol.) Triton X-100 for 3 min and incubated for 30 min in 20% (wt/vol.) Aquablock (East Coast Biologics, North Berwick, MA, USA) for blocking. Antibodies to the following antigens were used: guinea pig anti-human insulin (1:200; Abcam, Cambridge, MA, USA), rabbit anti-glucagon (1:20; Millipore, Billerica, MA, USA), goat anti-vimentin (1:10; Sigma-Aldrich, St Louis, MO, USA), mouse anti-human CD31 (1:50; BD Biosciences, San Jose, CA, USA), rat anti-mouse CD31 (1:100; BD Biosciences) and mouse anti-hVEGF (1:100; Millipore). The sections with these first antibodies were incubated at 4°C overnight. The antigens were visualised using appropriate secondary antibodies conjugated with FITC, Cy3 (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), Alexa-fluor-488 and Alexa-fluor-568 (Invitrogen). Secondary antibodies were used at concentrations recommended by the manufacturer. Then, DAPI was added to the sections for nuclear staining. In this study, the second antibodies against human CD31 and mouse CD31 were labelled with the same fluorochrome. Islets were visualised on a fluorescence microscope and images were analysed using MetaMorph software (Molecular Devices, Sunnyvale, CA, USA).
Insulin-positive beta cell mass in the host liver (left lobe) at day 32 after treatment was measured as previously described .
Capillary density measurement
Intra-insular CD31-positive vessels visualised by immunohistochemistry were considered as capillaries. The capillaries were counted by the use of fluorescence microscopy at a magnification of ×200. At least two photomicrographs of the insulin-positive graft islets in the host liver were taken for each mouse. The area of each islet was measured with ImageJ software (National Institutes of Health, Bethesda, MA, USA). Pre-isolation human islets were assessed as a control. Capillary density was expressed as the number/mm2. The investigator reading the capillary density was blinded to treatment group. The vessel density of mouse liver was measured with a similar procedure.
Effect of hVEGF on endogenous pancreas
To investigate the effect of circulating hVEGF on endogenous pancreas, UTMD with VEGF plasmid to the liver was performed in STZ-induced diabetic mice. Normal mice and STZ-induced diabetic mice were used as controls. Non-fasting blood glucose levels were measured for 20 days. At day 20 after UTMD, the pancreas in each group was harvested and investigated with immunohistochemistry for insulin (beta cells) and CD31 (capillaries). Insulin-positive beta cell mass in endogenous pancreas was measured as previously described . The vessel density in the islets was measured as described above.
Data are expressed as means ± SEM. Statistical significance of the differences among the three groups was determined by ANOVA followed by Student’s t test with Bonferroni correction. Differences of the ratios among the three groups were analysed by Tukey’s honestly significant difference test.
Effect of UTMD on gene delivery to liver via the portal vein
Serum hVEGF level after UTMD
Our preliminary experiments using pCI-hVEGF in vitro proved that hVEGF protein was secreted from the transfected cells into culture media (data not shown). We evaluated hVEGF levels in the blood of the mice treated with UTMD. hVEGF was detected for 14 days after UTMD, although the levels were low (Fig. 2g). This might be because of the effect of dilution and decomposition by blood.
Organ specificity of gene delivery
Various organs were harvested from the mice 3 days after UTMD via the portal vein and the VEGF expression in each organ was examined by RT-PCR. VEGF was strongly expressed in the liver, whereas other organs showed no expression (Fig. 2h). The right kidney slightly expressed VEGF, probably because it was exposed to ultrasound because of its anatomical proximity to liver.
Effect of UTMD and hVEGF on liver
To examine whether local expression of VEGF could increase the vessels in liver or cause uncontrollable angiogenesis and liver growth, the vessel density in liver and the weight of liver (left lobe) were measured. There were no remarkable differences among the three groups (no-UTMD, GFP and VEGF, Fig. 3c, d).
Effect of circulating hVEGF on endogenous pancreas
As shown in Fig. 2, there was no hVEGF production in the pancreas after UTMD, but hVEGF was detected in serum. We investigated whether this low level of hVEGF could affect endogenous pancreatic beta cells and islet vasculature. VEGF gene delivery to liver did not influence blood glucose levels (Fig. 3e), endogenous beta cell mass and vessel density in islets (Fig. 3f–h) for STZ-induced diabetic mice.
hVEGF production in and around graft islets
Effect of induced hVEGF on islet function after transplantation
The rate of euglycaemia 30 days after treatment in the three groups
Glucose tolerance test
To further define the function of the transplanted islets, IPGTTs were performed at day 31 after transplantation. The 2 h blood glucose levels in both the no-UTMD and GFP groups were >11.1 mmol/l, whereas the VEGF group showed a normal pattern (Fig. 5f).
Intra-islet vascularisation and beta cell mass
Revascularisation to the transplanted islets is essential to improve their survival [17, 18, 19, 20, 21]. It was reported that pancreatic islet production of VEGF is critical for islet vascularisation and function . To promote the revascularisation of the transplanted islets, ex vivo transduction of islets with an adenoviral vector encoding VEGF has been examined with evidence of revascularisation and improved islet survival [3, 23, 24, 25]. However, viral gene therapy is associated with severe adverse events [7, 8]. Hydrodynamics-based delivery of naked pDNA showed a therapeutic effect ; nevertheless its procedure is clinically unsuitable. It was shown that simple injection of pDNA alone without hydrodynamic pressure is ineffective . At first, we tested the infusion of naked VEGF plasmids via the portal vein without microbubbles and ultrasound because this is the simplest method. However, the gene expression was not detected in liver (data not shown). Then, we examined our developed UTMD method, which has been shown to be highly effective without significant adverse bioeffects [9, 10, 11, 27, 28, 29, 30].
In this study, we employed a clinically relevant intraportal islet transplantation model. Our results provide evidence that the VEGF gene can be targeted non-invasively to the liver and the transplanted islets, and can modify the intra-islet microvasculature. Notably, this is the first evidence that non-invasive delivery of a transgene to the host liver has therapeutic potential and it can produce biological changes in the vascularisation of graft islets. Intraportal injection of microbubbles has advantages. First, we could directly deliver a highly concentrated pDNA to liver compared with i.v. injection that was shown more effective than intrahepatic delivery . Second, it could result in efficient gene delivery to both the graft islet and the surrounding tissues. Obviously this could increase total gene expression compared with gene delivery only to islets, and it would be another advantage of UTMD over ex vivo gene delivery. For the purpose, we used CMV promoter instead of rat insulin promoter, which was shown to be effective in specifically directing genes to islets by UTMD. Although the results of this study indicated that the adverse effect of UTMD to liver was minimal, we should carefully assess hepatic toxicity in larger animals and ultimately in humans.
The increased microvasculature in graft islets probably contributed to the improvement of efficacy of islet transplantation. However, considering the time course of graft islet loss and VEGF expression and the extent of revascularisation, there might be the possibility of another mechanism for the effect of VEGF on the improved engraftment. Further studies would be required to reveal it.
In this study, the capillary density increased in the transplanted islets 32 days after VEGF plasmid gene transfer. Nevertheless, induced hVEGF protein was detectable in the treated mice only for 14 days in blood and for 10 days in liver. We previously reported that VEGF delivery to heart in the rat by UTMD increased capillary density but a regression of capillary density to the baseline level was observed by day 30, probably because of the transient nature of the plasmid expression . We speculate that normal myocardium does not require an additional blood supply, so that the angiogenic vessels regress. In contrast, transplanted islets need a new blood supply, and 10–14 days of hVEGF stimulation might lead to further angiogenesis in transplanted islets. Since upregulated hVEGF production could lead to abnormal blood vessels and haemangiomas in islets , the transient VEGF expression of our method can also be an advantage.
The effect of VEGF gene delivery by our method on the native pancreas was very minor and negligible as UTMD was targeted to the liver. Therefore, islet neogenesis in native pancreas does not explain the mechanisms of improved islet function.
Ultrasonic microbubble destruction causes cavitation, thermal effects, microstreaming, free radical production and microcapillary ruptures [33, 34, 35, 36], which might activate endothelial cells or other cells. Indeed, slight inflammation and disruption of hepatocyte architecture were observed in both the GFP and VEGF groups (data not shown), although our data showed very little liver dysfunction after UTMD. Islet transplantation itself causes severe inflammation; therefore, the potential adverse bioeffects of microbubble destruction appear to be offset by the therapeutic effects of gene therapy in the present study. To prevent the inflammation associated with islet transplantation, a combined use of VEGF and anti-inflammation genes or drugs might be useful .
In conclusion, VEGF gene delivery by UTMD to the transplanted islets and the host liver led to increasing vessel density in the graft islets and improving graft function. Gene delivery by UTMD is safe and effective in improving islet transplantation.
This work was supported by NIH R01 HL072430-01 (P. A. Grayburn) and NIH 2P01 DK58398 (Newgard). This work was also partially supported by All Saints Health Foundation.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
- 12.Matsumoto S, Noguchi H, Shimoda M et al (2010) Seven consecutive successful clinical islet isolations with pancreatic ductal injection. Cell Transplant. doi:10.3727/096368909X481773