research article primo vascular system: an endothelial-to...

Research ArticlePrimo Vascular SystemAn Endothelial-to-Mesenchymal Potential TransitionalTissue Involved in Gastric Cancer Metastasis

An Ping1 Su Zhendong2 Qu Rongmei3 Dai Jingxing3 Chen Wei1 Zhou Zhongyin1

Luo Hesheng1 and Kwang-Sup Soh4

1Department of Gastroenterology Renmin Hospital of Wuhan University Wuhan 430070 China2Department of Biochemistry Nagoya University Graduate School of Medicine Nagoya 466-8550 Japan3Department of Anatomy Southern Medical University Guangzhou 510515 China4Nano Primo Research Center Advanced Institute of Convergence Technology Seoul National UniversitySuwon-si 443-270 Republic of Korea

Correspondence should be addressed to Luo Hesheng luohswhugmailcom and Kwang-Sup Soh kssoh1gmailcom

Received 3 October 2014 Revised 19 January 2015 Accepted 21 January 2015

Academic Editor Kuang C Lai

Copyright copy 2015 An Ping et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Gastric cancer is the fourth commonest cancer in the world and the second leading cause of cancer-related death Investigationof gastric cancer metastasis is one of the hottest and major focuses in cancer research Growing evidence manifested that primovascular system (PVS) is a new kind of circulatory system beyond vascular and lymphatic system Previous researches revealedthat PVS is a specific tissue between endothelium and mesenchyme and is involved in cancer especially in tumor metastasis andregeneration In current study we investigated the role of primo vessels in gastric cancer metastasis and its possible relationship tovascular vessels formation Our results indicated that primo vessels were involved in gastric cancer metastasis We observed bloodvessel-mediated metastasis primo vessel-mediated metastasis and an intermediate state between them We deduced that primovessels may be precursors of blood vessels These results possibly provided a thoroughly new theoretic development in cancermetastasis

1 Introduction

Gastric cancer is the fourthmost common cancer in theworldand the second leading cause of cancer-related death despiteits declined incidence over the last 50 years [1 2] More than80 of diagnoses occur at the middle to late stage of thedisease andmetastasis to remote organs frequently is detected[3] Investigation of gastric cancer metastasis is one of thehottest and major focuses in cancer research Generally themetastasis of gastric cancer has three patterns hematogenousmetastasis through vascular vessels lymphatic metastasisthrough lymphatic vessels and implantation metastasisRecently growing evidence manifested that primo vascularsystem (PVS) is a new kind of circulatory system beyond vas-cular and lymphatic system [4ndash6]The functional role of PVSin cancer especially in tumor metastasis and regenerationwas extensively investigated and recognized by more and

more researchers [7ndash10] Our previous studies manifestedthat primo vessels in the mesentery are a specific transitionalstructure between endothelium and mesenchyme [11] Wededuced that primo vessel cells may be a sort of specificpopulation that had potential capability of transition toendothelial cells or fibroblasts Therefore our current studycontinued to investigate the role of primo vessels in gastriccancer metastasis and its possible relationship to vascularvessels formation

2 Materials and Methods

21 Cell Line andCell Culture TheMKN-45 cell line a poorlydifferentiated human gastric cancer cell line was obtainedfrom Korean Cell Line Bank The cells were maintainedin RPMI 1640 (GIBCO CA) supplemented with 10 fetalbovine serum (GIBCO CA) 1 penicillinstreptomycin and

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 812354 8 pageshttpdxdoiorg1011552015812354

2 Evidence-Based Complementary and Alternative Medicine

GFP DAPI GFPDAPI

(a)

GFP DAPI GFPDAPI

(b)

Figure 1 Isolation of stable high GFP expressed MKN-45-GFP cells (a) The enhanced GFP4 expression plasmid was transfected MKN-45cells The brightest fluorescent cells were sorted and cloned by flow cytometry Stable high GFP-expression clones were isolated and culturedin the absence of G418 for 10 passages (b) Stable high GFP-expression MKN-45-GFP cells cultured in the absence of G418 for 20 passages

1 L-glutamine (Sigma-Aldrich St Louis MO) and culturedin a humidified incubator in an atmosphere of 5 CO2 at37∘C ForGFP gene transduction the enhancedGFP4 expres-sion plasmids (Clontech Palo Alto CA) were transfectedinto 90 to 95 confluent MKN-45 cells using Lipofec-tamine 2000 (Invitrogen CA) according to the instructionsof the manufacturer Transfected MKN-45 cells (MKN-45-GFP) were cultured into a selective medium that contained200mgmL of G418 (GIBCO CA) The level of G418 wasincreased to 1000mgmL stepwise The brightest fluorescentcells (above the 95 percentile) were sorted and cloned by flowcytometry High GFP-expression populations were isolatedwith cloning cylinders (Bel-Art Products Pequannock NJ)and cultured in the absence of G418 for over 10 passages toselect for stable expression of GFP

22 Mice Adult male nude mice (BALB-c-nunu aged 5ndash7 weeks weighing 15ndash20 g 119899 = 20) were obtained fromJung-Ang Laboratory Animal Company (Seoul Republic ofKorea) The animals were maintained in a barrier facility inracks filtered with high-efficiency particulate air filter All theanimal care and studies were approved by the institutionalcommittee on research animal care and were done accordingto the Korean Policy on Humane Care and Use of LaboratoryAnimals

23 Orthotopic Implantation in Stomach MKN-45-GFP cellswere collected at the log phase and injected subcutaneouslyinto the mice at 10702mL Six weeks later tumors wereharvested from the mice under anesthesia (by subcutaneousinjection of 01mL of solution of 004 Zoletil and 006

Rompun) and minced into small pieces (2 times 2 times 2mm3) inHBSS containing 100UmL penicillin and 100 120583gmL strep-tomycin (Figures 2(a)ndash2(c)) For implantation the mousestomach was gently exteriorized via a left-side upper abdom-inal incision and one small tissue pocket was formed in themiddle wall of the greater curvature usingmicroscissors Onetumor piece was placed into the pocket and closed with a6-0 surgical suture (Figures 2(d) and 2(e)) Animals werekept in a sterile environment All procedures of the operationdescribed above were performed with an X7 magnificationmicroscope (Olympus)

24 Intraoperative Imaging From 6 to 8 weeks after theorthotopic implantation abdomens of the tumor-bearingmice were incised Metastatic tumors and primo vascularsystem were carefully observed under the X7 magnificationmicroscopeMetastatic tumors and primo vessels with classicappearances of free thread-like and semitransparent struc-ture were captured with a CCD camera (Axiophot ZeissGermany) To detect the GFP fluorescence derived from thegastric cancer a Leica stereo fluorescence microscope LZ12(Leica Microsystems Inc Bannockburn IL) equipped witha mercury lamp power supply was used Under the LZ12microscope the GFP fluorescence showed sensitive whitecolor High resolution images of 1024 times 724 pixels werecaptured Images were processed for contrast and brightnessand analyzed with the use of Image Pro Plus 40 software(Media Cybernetics Silver Springs MD)

25 Phalloidin and DAPI Staining in Tissue SamplesAfter intraoperative imaging primo vessels together with

Evidence-Based Complementary and Alternative Medicine 3

(a) (b) (c) (d) (e)

(f) (g)

GFP DAPI GFPDAPI

(h)

(i)

Figure 2 Orthotopic models in vivo and stable high level expression of GFP in gastric cancer metastatic tumors in nude mice (a) and(b) 5 times 106 MKN-45-GFP cells were inoculated subcutaneously into the left flank of 5ndash7-week-old anesthetized nude mice 6 weeks latersubcutaneous tumor grew obviously (c) Subcutaneous tumor was harvested and incised into small fragments (d) and (e) Tumor pieces wereorthotopically implanted in the gastric wall with surgical suture (f) and (g) Under the LZ12 microscope gastric orthotopic tumors expressedstrong GFP fluorescence (h) GFP signals from subcutaneous tumor pieces were tested under fluorescence microscopy (i) 6 to 8 weeks afterthe orthotopic implantation gastric metastasis was investigated Detection of GFP signal confirmed the metastatic tumors (arrows) andmicrotumors or gastric cancer cells (arrowheads)

metastatic tumors were taken out carefully To furtheridentify that the primo vessels were involved in gastriccancer metastasis phalloidin (Invitrogen CA) and 410158406-di-amidino-2-phenylindole (DAPI Invitrogen CA) stainingwere performed as described previously [11] Staining

was analyzed independently by two investigators using afluorescence microscope (BX51 Olympus Japan)

26 HampE Staining of Metastatic Tumors and Primo VesselsSubsequently features of primo vessels and relatedmetastatic


(a) (b) (c)

(d) (e) (f)

Phalloidin PhalloidinDAPIDAPI

(g)

Figure 3 Primo vesselswere involved in gastric cancermetastasis (a)A free primo vessel (black arrow)with a thread-like and semitransparentappearance between tumors (b) Under the LZ12 microscope a tiny metastatic tumor (black arrow) was observed in the primo vessel (c)Magnification of (b) more microtumors or gastric cancer cells (yellow arrows) were detected (d) HE staining for the metastatic tumormediated by primo vessels (e) Magnification of framed area of (d) (Bar 100120583m) (f) Transversal section of the primo vessel embeddedwas stained by HampE (Bar 50 120583m) No vascular vessels or lymphatic vessels were observed in either metastatic tumors or primo vessels (g)Primo vessels involved in gastric cancer metastasis showed rod shape nuclei in a linear arrangement (white arrows) and positively expressedphalloidin

tumors were determined by hematoxylin and eosin (HampE)staining Briefly metastatic tumors and primo vessels werefixed in 10 neutral buffered formalin (NBF) (pH 76) at4∘C After dehydration and clearing all the samples wereembedded in paraffin and sectioned using a Vibratome Series1000 sectioning system (Technical Products International StLouis MO) for further HampE staining

3 Results and Discussion

31 Isolation of Stable High Level GFP-Expression MKN-45-GFP Cells In the past GFP has been used to detectmetastatic cells in freshly excised tissue samples [12ndash14] and

for studies of cell motility within primary tumors by invivo time-lapse confocal microscopy [15] Studies suggestedthat the use of GFP-expressing cells could improve in vivoinvestigations of the metastatic process Utilization of GFP asa cell label for in vivo experiments requires that cells shouldbe stably transfected and express GFP over a long term Thestable transfected MKN-45-GFP cells used in the presentstudy exhibited a stable and strikingly bright fluorescence(screened by flow cytometry) in the absence of selective agent(G418) after numerous passages (Figure 1) This indicates thesuitability and sensitivity of the MKN-45-GFP cells for longterm use in vivo


Primovessel

Primovessel

vessel

vesselVascular

Lymphatic

CD31

CD31

LYVI-1

LYVI-1

Merge

Merge

Merge

Merge

DAPI

DAPI

DAPI

DAPI

Figure 4 Primo vessels were distinct from vascular vessels and lymphatic vessels Weak CD31 fluorescence (an endothelium marker) wasobserved in primo vessels while strong CD31 expression was detected in vascular vessels Furthermore primo vessels negatively expressedLYVI-1 (a lymphatic endothelium marker)

32 Stable High GFP-Expression in Gastric Metastatic TumorsAfter subcutaneous inoculation of MKN-45-GFP cells innude mice tumors were harvested As shown in Figure 2(h)tumor pieces expressed high GFP fluorescence which furtherindicated the high quality of stably transfected MKN-45-GFP cells Then small tumor pieces were orthotopicallyimplanted into the gastric wall Sixndasheight weeks later metas-tasis in orthotopic implantationmice was investigated Underthe LZ12 microscope gastric orthotopic tumors expressedstrong GFP fluorescence (Figures 2(f) and 2(g)) Further-more images also showed that metastatic tumors espe-cially multiple micrometastatic tumors were detected clearly(Figure 2(i)) All these observations suggested successfulanimalmodels for gastric cancer orthotopic implantation andmetastasis investigations

33 Primo Vessels Involved in Gastric Cancer MetastasisNext we investigated the role of primo vascular system in

gastric cancer metastasis We discovered that in orthotopicimplantation mice between the orthotopic tumors and themetastatic tumors there are free thread-like and semitrans-parent structures that possess the basic features of primovessels (Figure 3(a)) Under the LZ12 microscope besidesthe fact that metastatic tumors with GFP fluorescence weredistinctly detected tiny tumors in primo vessels were clearlyobserved (Figure 3(b)) Magnified images revealed multiplemicrotumors in primo vessels (Figure 3(c)) More impor-tantly in HampE staining no vascular vessels or lymphaticvessels were observed in metastatic tumors (Figures 3(d) and3(e)) and primo vessels (Figure 3(f)) Phalloidin and DAPIstaining revealed that these primo vessels expressed a lineararrangement of rod shape nuclei and positive phalloidinexpression which conformed with the characters of primovessels (Figure 3(g)) Accordantwith our previous studies thestructure of primo vessels was obviously distinct from that ofvascular vessels and lymphatic vessels


(a) (b)

(c) (d)

(e)

Figure 5 Vascular vessel-mediated metastasis (a) In vascular vessel-mediated metastasis metastatic tumors showed orange color and bloodvessels were observed (black arrows) (b) Under the LZ12 microscope metastatic tumor expressed bright GFP fluorescence and vascularvessels were obvious (yellow arrows) (c) Microtumors in the stalk of metastatic tumors were observed HampE staining revealed blood vesselsin the stalk (d) and metastatic tumors (e)

We further studied the biologic features of primo vesselsinvolved in gastric metastasis Our results indicated that con-trast to vascular vessels and lymphatic vessels primo vesselsexpressed weak CD31 (a endothelium marker) and negativeLYVE-1 (a lymphatic endothelium marker) (Figure 4) Thesedata supported our previous opinion that primo vesselsbelong to a type of endothelial-to-mesenchymal transitionaltissues [11] and suggested them as a new metastatic pathwayin gastric metastasis This is another new finding and proofthat revealed the role of primo vascular system in cancermetastasis

Compared with metastasis mediated by vascular vesselswe observed an interesting phenomenon that the bright-ness of GFP-tagged metastatic tumors was much differ-ent between primo vessel-mediated metastasis and vascularvessel-mediated ones In vascular vessel-mediatedmetastasistumors appeared yellow color (Figure 5(a)) and expressedmuch brighter GFP fluorescence (Figure 5(b)) while primovessel-mediated metastatic tumors showed whiter color (Fig-ure 2(a)) but weaker GFP signal (Figure 3(b)) In highmagnified images microtumors in the stalk of metastatictumors also were observed (Figure 5(c)) HampE staining


(a) (b)

(c) (d)

Figure 6 Intermediate state of metastasis between primo vessel-mediated metastasis and vascular vessel-mediated metastasis (a)Intermediate metastatic tumors between primo vessel-mediated metastasis and blood vessel-mediated metastasis were detected Half of thetumor showed white color (black arrow) and the other part showed orange color (black arrowhead) (b) Lifting the metastatic tumor in(a) a free semitransparent and thread-like structure was detected (black arrow) (c) Magnification of metastatic tumor red blood vesselsand capillaries were obvious (black arrow) (d) Under the LZ12 microscope high GFP fluorescence was expressed in the proximal part ofmetastatic tumor (yellow arrow) while lower GFP fluorescence was expressed in the distal part (yellow arrowhead)

confirmed vascular vessels in the stalks andmetastatic tumorsin vascular vessel-mediated metastasis (Figures 5(d) and5(e)) Interestingly intermediate metastatic tumors betweenthese two states were detected As shown in Figure 6 halfof the tumor showed white color and the other part showedorange color Because the cancer cells employed in ourexperiment were transfected by GFP plasmids we deducedthat the different color indicated different amount of cancercells in metastasis tumors Further fluorescence detectionrevealed the different GFP signals between these two parts(Figure 6(d))

Our previous study has revealed that primo vessel cellsbelong to a population between endothelium and mes-enchyme [11] Together with our current observations of thethree states in gastric cancer metastasis we hypothesizedthat primo vessels may be the precursor for vascular vesselsThese results possibly provided a thoroughly new theoreticdevelopment in cancer metastasis Much more work shouldbe done to further reveal the relationship between primovessels and vascular vessels We will continue our studiesin disclosing the role of primo vascular system in cancermetastasis

4 Conclusions

In conclusion our research demonstrated that primo vesselsare involved in gastric cancer metastasis and may be apotential precursor of vascular vessels These results possiblyprovided a thoroughly new theoretic development in cancermetastasis

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported in part by a grant from NationalNatural Science Foundation of China (81302131) andNatural Science Foundation of Hubei Province China(2012FKB04432)

References

[1] J-Y Deng and H Liang ldquoClinical significance of lymph nodemetastasis in gastric cancerrdquoWorld Journal of Gastroenterologyvol 20 no 14 pp 3967ndash3975 2014


[2] X Jin Z Zhu and Y Shi ldquoMetastasis mechanism andgeneprotein expression in gastric cancer with distant organsmetastasisrdquo Bulletin du Cancer vol 101 no 1 pp E1ndashE12 2014

[3] Y Kodera K Fujitani N Fukushima et al ldquoSurgical resectionof hepatic metastasis from gastric cancer a review and newrecommendation in the Japanese gastric cancer treatmentguidelinesrdquo Gastric Cancer vol 17 no 2 pp 206ndash212 2014

[4] R R Markwald T P Fitzharris and F J Manasek ldquoStructuraldevelopment of endocardial cushionsrdquoTheAmerican Journal ofAnatomy vol 148 no 1 pp 85ndash119 1977

[5] M Stefanov M Potroz J Kim J Lim R Cha and M HNam ldquoThe primo vascular system as a new anatomical systemrdquoJournal of Acupuncture and Meridian Studies vol 6 no 6 pp331ndash338 2013

[6] P An J Dai Z Su et al ldquoPutative primo-vascular system inmesentery of ratsrdquo Journal of Acupuncture andMeridian Studiesvol 3 no 4 pp 232ndash240 2010

[7] J S Yoo M H Ayati H B Kim W-B Zhang and K-S Soh ldquoCharacterization of the primo-vascular system inthe abdominal cavity of lung cancer mouse model and itsdifferences from the lymphatic systemrdquo PLoS ONE vol 5 no4 Article ID e9940 2010

[8] K A Kang C Maldonado G Perez-Aradia P An and K-SSoh ldquoPrimo vascular system and its potential role in cancermetastasisrdquo Advances in Experimental Medicine and Biologyvol 789 pp 289ndash296 2013

[9] H G Kim ldquoFormative research on the primo vascular systemand acceptance by the Korean scientific community the gapbetween creative basic science and practical convergence tech-nologyrdquo Journal of Acupuncture andMeridian Studies vol 6 no6 pp 319ndash330 2013

[10] B C Lee W J Akers X Jing M I Perez and Y Ryu ldquoPrimovascular system past present and futurerdquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID240168 2 pages 2013

[11] A Ping S Zhendong D Jingxing and et al ldquoDiscovery ofendothelium and mesenchymal properties of primo vessels inthemesenteryrdquo Evidence-Based Complementary and AlternativeMedicine vol 2013 Article ID 205951 10 pages 2013

[12] T Chishima Y Miyagi X Wang et al ldquoVisualization of themetastatic process by green fluorescent protein expressionrdquoAnticancer Research vol 17 no 4 pp 2377ndash2384 1997

[13] M Yang S Hasegawa P Jiang et al ldquoWidespread skeletalmetastatic potential of human lung cancer revealed by greenfluorescent protein expressionrdquo Cancer Research vol 58 no 19pp 4217ndash4221 1998

[14] T-T Yang P Sinai G Green et al ldquoImproved fluorescence anddual color detection with enhanced blue and green variantsof the green fluorescent proteinrdquo The Journal of BiologicalChemistry vol 273 no 14 pp 8212ndash8216 1998

[15] M Kneen J Farinas Y Li and A S Verkman ldquoGreenfluorescent protein as a noninvasive intracellular pH indicatorrdquoBiophysical Journal vol 74 no 3 pp 1591ndash1599 1998

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


GFP DAPI GFPDAPI

(a)

GFP DAPI GFPDAPI

(b)

Figure 1 Isolation of stable high GFP expressed MKN-45-GFP cells (a) The enhanced GFP4 expression plasmid was transfected MKN-45cells The brightest fluorescent cells were sorted and cloned by flow cytometry Stable high GFP-expression clones were isolated and culturedin the absence of G418 for 10 passages (b) Stable high GFP-expression MKN-45-GFP cells cultured in the absence of G418 for 20 passages

1 L-glutamine (Sigma-Aldrich St Louis MO) and culturedin a humidified incubator in an atmosphere of 5 CO2 at37∘C ForGFP gene transduction the enhancedGFP4 expres-sion plasmids (Clontech Palo Alto CA) were transfectedinto 90 to 95 confluent MKN-45 cells using Lipofec-tamine 2000 (Invitrogen CA) according to the instructionsof the manufacturer Transfected MKN-45 cells (MKN-45-GFP) were cultured into a selective medium that contained200mgmL of G418 (GIBCO CA) The level of G418 wasincreased to 1000mgmL stepwise The brightest fluorescentcells (above the 95 percentile) were sorted and cloned by flowcytometry High GFP-expression populations were isolatedwith cloning cylinders (Bel-Art Products Pequannock NJ)and cultured in the absence of G418 for over 10 passages toselect for stable expression of GFP

22 Mice Adult male nude mice (BALB-c-nunu aged 5ndash7 weeks weighing 15ndash20 g 119899 = 20) were obtained fromJung-Ang Laboratory Animal Company (Seoul Republic ofKorea) The animals were maintained in a barrier facility inracks filtered with high-efficiency particulate air filter All theanimal care and studies were approved by the institutionalcommittee on research animal care and were done accordingto the Korean Policy on Humane Care and Use of LaboratoryAnimals

23 Orthotopic Implantation in Stomach MKN-45-GFP cellswere collected at the log phase and injected subcutaneouslyinto the mice at 10702mL Six weeks later tumors wereharvested from the mice under anesthesia (by subcutaneousinjection of 01mL of solution of 004 Zoletil and 006

Rompun) and minced into small pieces (2 times 2 times 2mm3) inHBSS containing 100UmL penicillin and 100 120583gmL strep-tomycin (Figures 2(a)ndash2(c)) For implantation the mousestomach was gently exteriorized via a left-side upper abdom-inal incision and one small tissue pocket was formed in themiddle wall of the greater curvature usingmicroscissors Onetumor piece was placed into the pocket and closed with a6-0 surgical suture (Figures 2(d) and 2(e)) Animals werekept in a sterile environment All procedures of the operationdescribed above were performed with an X7 magnificationmicroscope (Olympus)

24 Intraoperative Imaging From 6 to 8 weeks after theorthotopic implantation abdomens of the tumor-bearingmice were incised Metastatic tumors and primo vascularsystem were carefully observed under the X7 magnificationmicroscopeMetastatic tumors and primo vessels with classicappearances of free thread-like and semitransparent struc-ture were captured with a CCD camera (Axiophot ZeissGermany) To detect the GFP fluorescence derived from thegastric cancer a Leica stereo fluorescence microscope LZ12(Leica Microsystems Inc Bannockburn IL) equipped witha mercury lamp power supply was used Under the LZ12microscope the GFP fluorescence showed sensitive whitecolor High resolution images of 1024 times 724 pixels werecaptured Images were processed for contrast and brightnessand analyzed with the use of Image Pro Plus 40 software(Media Cybernetics Silver Springs MD)

25 Phalloidin and DAPI Staining in Tissue SamplesAfter intraoperative imaging primo vessels together with


(a) (b) (c) (d) (e)

(f) (g)

GFP DAPI GFPDAPI

(h)

(i)






(a) (b) (c)

(d) (e) (f)


(g)







Primovessel

Primovessel

vessel

vesselVascular

Lymphatic

CD31

CD31

LYVI-1

LYVI-1

Merge

Merge

Merge

Merge

DAPI

DAPI

DAPI

DAPI






(a) (b)

(c) (d)

(e)





(a) (b)

(c) (d)




4 Conclusions




Acknowledgments


References






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b) (c) (d) (e)

(f) (g)

GFP DAPI GFPDAPI

(h)

(i)






(a) (b) (c)

(d) (e) (f)


(g)







Primovessel

Primovessel

vessel

vesselVascular

Lymphatic

CD31

CD31

LYVI-1

LYVI-1

Merge

Merge

Merge

Merge

DAPI

DAPI

DAPI

DAPI






(a) (b)

(c) (d)

(e)





(a) (b)

(c) (d)




4 Conclusions




Acknowledgments


References






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b) (c)

(d) (e) (f)


(g)







Primovessel

Primovessel

vessel

vesselVascular

Lymphatic

CD31

CD31

LYVI-1

LYVI-1

Merge

Merge

Merge

Merge

DAPI

DAPI

DAPI

DAPI






(a) (b)

(c) (d)

(e)





(a) (b)

(c) (d)




4 Conclusions




Acknowledgments


References






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Primovessel

Primovessel

vessel

vesselVascular

Lymphatic

CD31

CD31

LYVI-1

LYVI-1

Merge

Merge

Merge

Merge

DAPI

DAPI

DAPI

DAPI






(a) (b)

(c) (d)

(e)





(a) (b)

(c) (d)




4 Conclusions




Acknowledgments


References






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)

(e)





(a) (b)

(c) (d)




4 Conclusions




Acknowledgments


References






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)




4 Conclusions




Acknowledgments


References






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article primo vascular system: an endothelial-to...

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