In ovo administration of human recombinant leptin shows dose dependent angiogenic effect on chicken chorioallantoic membrane
© Manjunathan and Ragunathan. 2015
Received: 18 February 2015
Accepted: 2 June 2015
Published: 10 June 2015
Leptin, the cytokine produced by white adipose tissue is known to regulate food energy homeostasis through its hypothalamic receptor. In vitro studies have demonstrated that leptin plays a major role in angiogenesis through binding to the receptor Ob-R present on ECs by stimulating and initiating new capillary like structures from ECs. Various in vivo studies indicate that leptin has diverse effect on angiogenesis. A few reports have showed that leptin exerts pro angiogenic effects while some suggested that it has antiangiogenic potential. It is theoretically highly important to understand the effect of leptin on angiogenesis to use as a therapeutic molecule in various angiogenesis related pathological conditions. Chicken chorio allantoic membrane (CAM) on 9th day of incubation was incubated with 1, 3 and 5 μg concentration of HRL for 72 h using gelatin sponge. Images where taken after every 24 h of incubation and analysed with Angioguant software. The treated area was observed under microscope and histological evaluation was performed for the same. Tissue thickness was calculated morphometrically from haematoxylin and eosin stained cross sections. Reverse transcriptase PCR and immunohistochemistry were also performed to study the gene and protein level expression of angiogenic molecules.
HRL has the ability to induce new vessel formation at the treated area and growth of the newly formed vessels and cellular morphological changes occur in a dose dependent manner. Increase in the tissue thickness at the treated area is suggestive of initiation of new capillary like structures. Elevated mRNA and protein level expression of VEGF165 and MMP2 along with the activation of ECs as demonstrated by the presence of CD34 expression supports the neovascularization potential of HRL.
Angiogenic potential of HRL depends on the concentration and time of incubation and is involved in the activation of ECs along with the major interaction of VEGF 165 and MMP2. It is also observed that 3 μg of HRL exhibits maximum angiogenic potential at 72 h of incubation. Thus our data suggest that dose dependent angiogenic potential HRL could provide a novel role in angiogenic dependent therapeutics such as ischemia and wound healing conditions.
KeywordsAngiogenesis HRL, Human Recombinant Leptin CAM, chorioallantoic membrane ECs, endothelial cells VEGF, vascular endothelial growth factor MMP, matrix metalloproteinase Blood vessels CD34
The Ob-gene product leptin is 16 kDa peptide hormone, produced mainly by white adipose tissue is proposed to play a key role in the regulation of body weight and thermogenesis through the receptor found in the hypothalamus . Accumulating evidences from in vitro and in vivo assays suggest that leptin promote endothelial cell (EC) proliferation and survival in favour of angiogenesis [2, 3]. In vivo and in vitro results from Hyung et al. study indicates that angiogenic potential of leptin is mediated by matrix metalloproteinase (MMPs) . However, the actual mechanism behind the involvement of matrix metalloproteinase in leptin mediated angiogenesis is not clear.
The effect of leptin on angiogenesis is still not understood as some reports have suggested its potential to induce angiogenesis while some have indicated of its anti angiogenic effect [5–9]. Recently it has been reported that in ovo administration of leptin inhibited angiogenesis on chicken chorio allantoic membrane (CAM) . Leptin has been administered as a therapeutic molecule in various angiogenesis related pathological conditions especially in wound healing . However a quantitative in vivo evaluation on the effect of leptin on angiogenesis is important because of its therapeutic application potential in the pathology of angiogenesis dependent conditions. Therefore in the present study we examined the angiogenic potential of human recombinant leptin (HRL) using well vascularized CAM of developing chicken embryo (Gallus gallus domesticus). The advantage of CAM assay is that it is a highly vascularized structure with potential growth and this in ovo vivo system is highly useful to understand the physiological angiogenesis and hence widely used for the screening of various compounds for their angiogenic activity .
In the present study we analysed and compared the angiogenic ability of HRL of varying concentrations such as 1, 3 and 5 μg for an incubation period of 72 h using late CAM. Gelatin sponges soaked with leptin were placed on the membrane at 9th day of post incubation so as to allow slow delivery of the chemical at the treated area with less or no inflammation [13, 14]. The ability of HRL to induce new vessel growth at the treated area is directly visualized from the CAM images taken at different time period of incubation and growth of these vessels measured with Image J and Angioquant MATLAB softwares [14–16]. Angiogenic response of HRL at the treated area is further analysed in detail from the histological sections. We also examined the expression of major angiogenic molecules at the molecular and protein level to understand the involvement of these factors on HRL induced angiogenesis. Our findings suggest that the potential of HRL to induce angiogenesis depends on various physiological factors especially dose and time of incubation. The result have demonstrated that HRL favours neovascularization through sprouting of vessels which is accelerated by the expression of VEGF165 and MMP2 in a dose dependent way in chicken CAM vasculature.
Fertilized white leghorn chicken eggs were purchased from Tamil Nadu Poultry Research Station, Madras Veterinary University, Nandanam, Tamil Nadu, India. Gelatin sponges purchased from Jhonson & Jhonson Pvt Lmtd, India, Paraffin film and wax were purchased from Sigma, Aldrich, USA. Haematoxylin and Eosin stain purchased from Medox, India and Human recombinant leptin from MP Biomedicals, Inc. France. TRIzol reagent, agarose and EtBr were from Sigma, Aldrich, USA. ImProm-11™ Reverse Transcriptase kit and GoTaq Green Master Mix PCR amplification kit were from Promega, USA, Oligo (dt) of length 18-meres from eurofins, mwg operon, Germany, Random hexamers from MP Biomedicals, USA. All primers were purchased from Bioserve, India. DNA ladders purchased from Invitrogen, USA. DAB system purchased from Bangalore Genei, India. CD34 antibody (Endothelial Cell Marker, Cluster designation 34) from US Biological, USA and VEGF A from CALBIOCHEM, EMD. Bradford reagent and FITC (Goat ant-rabbit IgG were from Bangalore Genei, India. SDS-PAGE Standards marker was from BIO-RAD, CANADA and gelatin from Medox, India. Rabbit polyclonal MMP2 is a kind gift from Dr. Li Haiqing, MD, Ph.D, −Technology transfer specialist, National Cancer Institute, Rockville, USA. Unless otherwise specified all other common reagents and chemicals were purchased from Sigma, Aldrich, USA.
The in ovo CAM model
Fertilized white leghorn chicken eggs weighing 50 ± 2 g, were incubated at 37 °C in a ‘humidified atmosphere (>60 % relative humidity) as per the protocol described in Hen’s Egg Test - Chorioallantoic Membrane (HET-CAM) method adapted from ZEBET (The German Centre for the Documentation and validation of Alternative Methods, Republic of Germany). At day 3 of post incubation, 2 to 3 ml of albumin was withdrawn, using a 21 gauge needle, through the large blunt edge of the egg in order to minimize the adhesion of the shell membrane with CAM. A square window of 2 cm2 was opened in the egg shell and sealed with paraffin film to prevent dehydration and the eggs were incubated further. At day 9 of post incubation, gelatin sponges of size of 1 mm3 were placed on top of the growing CAM under sterile condition [14, 17] and were soaked with 15 μl volume of 1, 3 and 5 μg concentration of HRL. Control eggs were incubated with 1X PBS (pH-7.3). The window was closed with a transparent adhesive tape and eggs were incubated for 72 h until it reached post incubation day 12. CAM were photographed at 0, 24, 48 and 72 h using Canon digital camera and images were analysed with Image J and Angioquant Toolbox, MATLAB 6.5 software to measure total length and size of the blood vessels (micrometre) from the area of treatment.
Light microscope analysis
After 72 h of incubation the area of the CAM treated with HRL was detached carefully. The excised membrane was kept on glass slides and images were taken using light microscope both at 4 and 10× magnification to view the growth of capillaries .
After 72 h of incubation area of the CAM treated with HRL was flooded with Bouin’s fixative solution. Around 1 cm2 of the membrane around the treated area was removed carefully using forceps and surgical scissors and dehydrated through graded series of alcohol (50 %, 70 %, 90 % and absolute) and embedded in paraffin wax. Vertical cross tissue sections (7 μm in thickness) were taken using Rotary Microtome (Weswicox, Japan). Sections were treated with alcohol in ascending order (absolute, 90 %, 70 %, and 50 %) and cleared with xylene before staining with haematoxylin and eosin. After mounting with DPX, the histological sections were observed under light microscope at 40× magnification for qualitative assessment and images were recorded using Nikon Camera attached with light microscope at 10× magnification .
Morphometric analysis of CAM tissue thickness (DCAM)
Thickness of the CAM for all the groups treated with HRL were measured from haematoxylin and eosin stained vertical cross sections using a calibrated objective at 40× magnification with 10 × 10 calibrated grid at 10× ocular. Distances between the chorionic and allantoic epithelial layers were measured in micrometre at 6 different locations from the same sample and is repeated for six serial cross sections of the same. Average tissue thickness was calculated from each tissue sample of the same and obtained a mean DCAM thickness [14, 19].
Semi-Quantitative Reverse Transcriptase–Polymerase Chain Reaction (RT-PCR)
Total RNA was isolated from CAM treated with HRL using TRIzol reagent (100 mg/1 ml). The quantity and the purity of the isolated RNA were checked using UV-visible spectrophotometer. cDNA of 20 μl volume was synthesized using ImProm-11™ Reverse Transcriptase kit with Oligo (dt) of length 18-meres and random hexamers. PCR amplification was performed using GoTaq Green Master Mix kit and changes in the level of mRNA expression of VEGF165 , VEGF121 , MMP2 , MMP9  and GAPDH  were evaluated using PCR with 100 pico moles of specific primers. The relative expression level of each mRNA transcript was normalized with that of control. PCR products were subjected to electrophoresis.
CAM tissues at the treated area from each group were homogenized (100 mg/ml) using Tris buffer (0.5 M Tris–HCl (pH-6.8), 10 % SDS, glycerol and 0.01 % bromophenol blue) and centrifuged at 12,000 rpm/4 °C/10 min. The concentration of protein from the supernatant was determined using Bradford reagent and the gelatinase activity was examined on a 10 % SDS-PAGE electrophoresis containing 1.0 mg/ml of gelatin. Protein samples of 25 μg/40 μl were loaded per well along with 20 μl pertained SDS-PAGE Standards marker. After electrophoresis, the gels were washed with 2.5 % of Triton-X-100 and incubated in digestion buffer (50 mM Tris HCl-pH-7.5,100 mM CaCl2, 1 μM ZnCl2, 1 % Triton X-100, 0.02 % NaN3-100 ml) for 16 to 18 h at 37 °C with gentle agitation. The gel was stained with staining solution (0.05 % (w/v, Coomassie blue in 50 % methanol and 10 % acetic acid) for 1 h and de stained with methanol/acetic acid mixture. Gelatinase activity of MMP2 was detected as clear white bands against background. The gels were scanned and images were recorded using BIO-RAD Calibrated Densitometer Software (GS 800, USA). The density of the bands was calculated with PD Quest Advances Software and normalized with control value .
Three different concentration namely 1, 3 and 5 μg of HRL have been used of which 3 μg of HRL yielded maximum angiogenic response when compared to other two concentrations. The deparafinized and dehydrated CAM of 5 μM thickness after treating with 3 μg of HRL was allowed to undergo antigen retrieval process using Sodium Citrate (10 mM-pH 6.0) in a microwave oven for 20 min followed by washing in DDH2O for 3X5 min in 1X PBS (pH 7.3). Normal Goat Serum Blocking Solution (2 % goat serum,1 % BSA, 0.1 % cold fish skin gelatin, 0.1 % Triton X-100, 0.05 % Tween- 20, 0.05 % Sodium Azide, 0.01 M PBS (pH 7.2) of 50 to 75 μl was added immediately on the sections and incubated for 1 h in a humidified chamber. After washing with 1X PBS, primary antibodies of MMP2 (1:200), VEGF (VEGF A) (1:100) and CD34 (1:200) diluted in blocking serum were applied on the sections and after overnight incubation rinsed with 1X PBS with 0.05 % of Tween-20. Diluted FITC (Goat ant-rabbit IgG) and HRP (both Goat anti-rabbit and Goat anti- mouse IgG) secondary antibodies of 1:40 dilution was applied for 1 h according to manufacturer’s instruction. For HRP conjugated secondary antibodies DAB system was used for colour development. The slides were finally counterstained with Mayer’s haematoxylin and mounted with 90 % of glycerol. For HRP conjugated system the images were recorded using light microscope and for FITC conjugation BX51 Olympus Fluorescence Microscope at a wavelength of 515 nm with ASI FISH View 5.5 software at 40× magnification .
Data analysis and statistical analysis
All experiments were independently performed in triplicate. Data were analysed using one way ANOVA analysis of variance test and Tukey post hoc test as appropriate (Sigma stat 2.0). Data were expressed as means + S.E.M and P-values of *p = < 0.001 and #p = 0.001 were selected for showing statistically significant difference.
Human recombinant leptin induces neo vascularization - visual assessment of new blood vessel formation on CAM vascular bed and its growth
From the above observation it can be inferred that, both 3 and 5 μg concentration of HRL has the ability to induce angiogenesis through new capillary formation at 72 h of incubation. In order to conform this and also to arrive at the optimum working concentration for HRL we measured the growth of the vessels in terms of its length and size, from the images taken at 0, 24, 48 and 72 h of incubation with Image J and Angioquant MATLAB software. This software is specially made for quantifying angiogenesis using in vitro assays and has been employed for CAM with some modifications using Image J software [11, 13]. Of the three different concentrations, used 3 μg of HRL demonstrates a significant increase in the vessel length (Fig. 1b. b) and size (Fig. 1b. b) at 48 h of incubation which is 1.99 fo1d greater than that of control (#p = 0.001, *p = <0.001). At 72 h of incubation both 3 and 5 μg of HRL induces a significant increase in the vessel length and size (*p = <0.001) and of these two, 3 μg of leptin shows 2.1 fold increases in vessel growth which is higher than that of 5 μg that showed which has only 1.8 fold increase. HRL of 1 μg also shows angiogenic potential with increase in the vessel growth up to 1.5 fold, the value is not significant when compared to control that has 1.2 fold increase in the vessel growth from 0 to 72 h of incubation. Thus it is obvious that HRL can induce new vessel formation at the treated area in a dose dependent manner with 3 μg concentration having maximum angiogenic response.
Human recombinant leptin initiate growth of vessels by means of sprouting – visualising the morphology of blood vessels on CAM vascular bed
Human recombinant leptin induces morphological changes leading to neo vascularization – histological observation of the CAM vasculature
Human recombinant leptin increases the size of tissue by means of inducing new vessel growth. Quantitative measurement of the angiogenic potential of human recombinant leptin by morphometric measurement of CAM tissue thickness (DCAM)
Angiogenic potential of human recombinant leptin depends on the activation of major angiogenic growth factors - molecular profiling of VEGF 165, VEGF 121, bFGF2, MMP2 and MMP9
Human recombinant leptin accelerates the gelatinase activity of MMP2
Human recombinant leptin enhances activation of endothelial cells
Angiogenic potential of human recombinant leptin mainly depends on the activation of VEGF A and MMP2 – protein expression
Angiogenesis is defined as the formation of new blood vessels from existing ones which plays an important role in many physiological and pathological events . Leptin, the Ob gene product is known to exert its biological activity through binding with it receptors termed Ob-R identified in hypothalamus and also in peripheral vascular tissue such as ECs [1, 29]. Reports from various in vivo analyses have suggested both angiogenic and anti angiogenic property of leptin [3, 10]. But no reports have yielded conclusion results. Reports have also suggested that this dual function of leptin depends on multiple factors such as dose, time of incubation, mode of administration and to certain extent the type of species [3, 4, 8–10, 30].
In this context we analysed the angiogenic potential of HRL using in vivo CAM assay. The main advantage of the assay is that it can be used as a rapid method of determining the angiogenic responses because it is considered as an intermediate step between a single model (cell culture) and a more complex system (mammalian model). The other important advantage is that CAM can be used to score the tissue responses towards the angiogenic activity of bio materials accurately and are similar to that of mammalian model responses  and also helps in the maintenances of the test materials at the site of administration. To overcome this technical problem we used gelatin sponges for the delivery of the chemical which can held and adhere firmly to the CAM surface with no or less inflammatory reactions [13, 32].
In the present work we found that in ovo administration of HRL is able to induce neovascularization at the treated area in a dose dependent manner. Among those analysed concentration (1, 3 and 5 μg) only 3 μg of HRL shows significant angiogenic response as observed the increase in vessel growth at an early incubation period of 48 h. The results are in accordance with the earlier research report where the effect was prominent even at 24 h of incubation . But this shorter time period of incubation on CAM is considered as vasodilation period rather than compound effect . Hence in the present study we performed the experiments for 72 h to analyse the angiogenic potential of HRL because at this time point newly formed vessels will become stabilized. Interestingly we found that at 72 h of incubation both 3 and 5 μg of HRL shows significant angiogenic effect indicating that the angiogenic potential of HRL not only depends on the concentration but also on the duration of incubation. Microscopic analysis of the growth pattern of the vessels implicates that 3 μg of leptin is able to induce more sprouting of new vessels from the existing one when compared to 5 μg concentration. Thus, HRL has the potential to induce neovascularization in a dose dependent manner and that 3 μg concentration could be more appropriate resulting in earlier and stable angiogenesis.
Placing any foreign material onto CAM can develop inflammatory reaction which can cause secondary vasoproliferative response leading to false positive conclusion . To avoid this issue we have performed a thorough histological evaluation after adding HRL. Changes in the histological structure and altered cellular morphology of the treated area together indicated that HRL is able to initiate new blood vessel formation without local inflammation. HRL of 3 μg shows comparatively higher angiogenic responses by accelerating the formation of new vessel through sprouting from the major one. Also the angiogenic or antiangiogenic effect of compound can be effectively analysed by measuring the thickness of the CAM at the treated area as formation and growth of the new blood vessels will result in an increased tissue thickness at the stroma region. Any changes related to the growth at the stroma will push chorion and allantoic epithelium a part . In our study we found that 3 μg of HRL is able to exert maximum angiogenic response at the stroma region very near to chorionic epithelium rather than mesodermal layer by means of forming capillary like tubes and structures leading to increased tissue thickness.
Results from rat corneal assay as also from various in vitro analyses showed that angiogenic effect of leptin is mediated by VEGF  and is supported by matrix remodelling . Molecular profiling of VEGF and its isoforms such as VEGF165 and VEGF121 indicates that HRL induced angiogenesis mainly depends on the expression of VEGF165 rather than VEGF121. Increased mRNA level of MMPs especially MMP2 confirmed the major role of MMP2 in matrix remodelling during HRL induced angiogenesis and is in agreement with the earlier report [29, 34, 35]. Various reports have suggested that the physiological functions of leptin get initiated once it binds to its receptor Ob-R present in various cellular systems. During angiogenesis leptin is shown to exert its activity by binding with Ob-R receptor present on ECs followed by up regulation of the downstream signalling . Here also it is possible that the angiogenic response of HRL on CAM is mediated by the receptor Ob-R considering the fact that human and chicken leptin receptor has 62 % of homology and the structural domains of the receptor are highly conserved in both . The present data suggests that 3 μg of HRL has more angiogenic potential by inducing the increased mRNA level expression of VEGF165, VEGF121, bFGF2, MMP2 and MMP9 and also the protein level expression of VEGF165 and MMP2. Thus of HRL could up regulate the expression of VEGF165 and MMP2 to induce neovascularization resulting in the observed sprouting at the area of treatment.
Activation of EC is considered as one of the most important and preliminary step in the cascade of angiogenesis . In vitro analysis suggested that leptin could initiate the activation of ECs to form tube like structures while inducing angiogenesis . In this work we also analysed the potential effect of HRL on the activation of ECs by studying the immuno localization of CD34 expression on CAM ECs. Presence of more activated ECs at the blood sinus region along with coiled like structures formation of ECs at the sinus area together highly support the ability of HRL to activate ECs during angiogenesis. Presences of elongated ECs at the lumen of the vessel also indicates that 3 μg of HRL could be the optimum concentration that can initiate the formation of new capillary like structures which is remarkable at the first phase of sprouting angiogenic process.
The present work demonstrates that HRL has the potential to induce neovascularization by means of sprouting at the treated area. Expression of CD34 on activated ECs indicates that HRL can initiate the activation ECs and could favour the formation of tube like structure from the pool of ECs. Increased tissue thickness and altered cellular morphology of CAM with HRL treatment supports dose dependent angiogenic ability of HRL. It was also observed that HRL induced angiogenesis mainly depends on the activation of VEGF165 and MMP2. Direct visualization and growth of newly formed vessels at the treated area demonstrates that the angiogenic ability especially sprouting effect of HRL depends on the concentration and time of incubation. It is found that 3 μg of HRL exhibits significant angiogenic response at 72 h of incubation. Altogether our findings suggest that HRL could be a useful angiogenic therapeutic molecule and can be more effectively used in the future especially for the treatment of ischemic disorders and wound healing which requires new vessel formation.
We would like to thank Dr. Shanthi, MBBS, MD (Pathology), Department of Pathology for immunohistochemical study and Mr. Thankaraj for microscope technical assistant. We would like to thank Dr. Li Haiqing, MD, Ph.D, −Technology transfer specialist, National Cancer Institute, Rockville, USA for the kind gift of Rabbit polyclonal MMP2 antibody. We thank the funding agency, University Grant commission of India for giving merit fellowship and also for supporting the department under SAP program.
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