VASCULAR BIOLOGY

Vascular endothelial growth factor-D transgenic mice show enhanced bloodcapillary density, improved postischemic muscle regeneration, and increasedsusceptibility to tumor formationAnna-Mari Karkkainen,1 Antti Kotimaa,1 Jenni Huusko,1 Ivana Kholova,1 Suvi Elina Heinonen,1 Anna Stefanska,1

Marike Hinke Dijkstra,1 Hanna Purhonen,1 Eveliina Hamalainen,1 Petri Ilmari Makinen,1 Mikko Petri Turunen,1 andSeppo Yla-Herttuala1

1A.I. Virtanen Institute, Department of Biotechnology and Molecular Medicine, University of Kuopio, Kuopio, Finland

Vascular endothelial growth factor-D(VEGF-D) has angiogenic and lym-phangiogenic activity, but its biologic rolehas remained unclear because knockoutmice showed no clear phenotype. Trans-genic (TG) mice expressing the matureform of human VEGF-D (hVEGF-D) wereproduced by lentiviral (LV) transgenesisusing the perivitelline injection method.Several viable founders showed a macro-scopically normal phenotype and thetransgene transmitted through germ line.Expression of hVEGF-D mRNA was high

in skeletal muscles, skin, pancreas, heart,and spleen. A significant increase wasfound in capillary density of skeletalmuscles and myocardium, whereas nochanges were observed in lymphatic cap-illary density. After induction of hindlimbischemia, the TG mice showed enhancedcapacity for muscle regeneration. How-ever, on aging the TG mice had signifi-cantly increased mortality from malig-nant tumors, of which half were breastadenocarcinomas characterized with theabsence of periductal muscle cells. Some

tumors metastasized into the lungs. Inaddition, lung and skin tumors werefound, but no blood- or lymphatic vessel–derived malignancies were detected. Weconclude that in mice hVEGF-D is anangiogenic factor associated with im-proved muscle regeneration after is-chemic injury but also with increasedincidence of tumor formation with a pref-erence for mammary gland tumors.(Blood. 2009;113:4468-4475)

Introduction

Vascular endothelial growth factors (VEGFs) are key mediators ofblood and lymphatic vessel formation during embryonic develop-ment and in adults. Blood vessel formation occurs either byvasculogenesis, when a new network of capillaries is formed fromendothelial precursor cells differentiating in situ, or by angiogen-esis, when new capillaries invade the organ by forming vascularsprouts that originate from pre-existing vessels.1 The lymphaticsystem comprises a separate vascular system that also permeatesmost organs. Lymphatic vessels are essential for immune surveil-lance, tissue fluid homeostasis, and fat absorption. Defects information or function of lymphatic vessels cause lymphedema, andlack of lymphatic vessels result in fluid accumulation, which causesprenatal death.2 Lymphangiogenesis occurs either by local de novodifferentiation of lymphatic endothelium from lymphangioblasts orby sprouting from preexisting lymphatic veins.3

Currently, 7 members have been identified in the VEFG family:VEGF-A, -B, -C, -D, -E (viral VEGF analogs), -F (snake venomVEGFs), and placental growth factor (PlGF). Of these, VEGF-Eand -F represent exogenous VEGFs.4

Several VEGF receptors (VEGFRs) have been identified for VEGFs:VEGFR-1 (flt-1; fms-induced tyrosine kinase receptor), VEGFR-2(KDR; kinase insert domain–containing receptor), and VEGFR-3 (flt-4)belong to the superfamily of receptor tyrosine kinases (RTKs). Theyshare a common structure, yet they bind VEGFs in distinct affinities andspecificities. In addition, Neuropilins 1 and 2 (Nrp-1, Nrp-2) bind some

specific VEGFs. Nrps are non-RTKs that are believed to serve ascoreceptors for certain VEGFs and their isoforms.5

VEGF-D (also known as c-fos–induced growth factor, FIGF) is asecreted growth factor consisting of a central VEGF-homology domain(VHD), receptor binding domains, and propeptides in both termini.VEGF-D is secreted into the extracellular space as a full-lengthVEGF-D homodimer.After secretion, proprotein convertases cleave theC- and N-terminal propeptides from the VHD to form the matureVEGF-D.6 The mature hVEGF-D binds to hVEGFR-2 and -3 withhigher affinities than the full-length unprocessed hVEGF-D. Numeri-cally, the mature hVEGF-D has approximately 290-fold higher affinityto hVEGFR-2 and approximately 40-fold higher affinity to hVEGFR-3than the unprocessed full-length hVEGF-D, which mainly binds tohVEGFR-3.7 In this article hVEGF-D refers to mature hVEGF-D�N�C.Cellular effects of hVEGF-D depend on the receptor and target cell type.Vascular endothelial cells express VEGFR-2, and the expression isup-regulated inter alia by angiogenesis.8 hVEGF-D exerts angiogeniceffects on binding to VEGFR-2, whereas when bound to VEGFR-3 onlymphatic endothelium it stimulates lymphangiogenesis. In addition toVEGFR-2 and -3, hVEGF-D is able to interact with Nrp-2, which has arole in lymphangiogenesis.5 VEGF-D probably possesses differingfunctions in different species; in mouse hVEGF-D binds to bothVEGFR-2 and -3, whereas mVEGF-D binds only to VEGFR-3.9,10

We used lentiviral (LV) perivitelline injection technique for thegeneration of transgenic (TG) mice and followed them up to

Submitted July 31, 2008; accepted November 14, 2008. Prepublished online asBlood First Edition paper, December 10, 2008; DOI 10.1182/blood-2008-07-171108.

An Inside Blood analysis of this article appears at the front of this issue.

The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 USC section 1734.

© 2009 by The American Society of Hematology

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generation F3. The LV transgenesis method is still rarely useddespite its versatility and advantages over traditional transgenesismethods.11 In this study, LV transgenesis was an effective methodfor producing TG offspring. We found that hVEGF-D TG miceshowed no major changes in lymphatic capillary density. Instead,they showed enhanced angiogenesis as well as improved muscleregeneration after injury. Unexpectedly, the TG mice possessed anincreased susceptibility to tumor formation.

Methods

Lentiviral vector construction

Vesicular stomatitis virus G-protein (VSV-G)–pseudotyped, third genera-tion LVs were constructed to directly express the mature form of hVEGF-Dunder the human phosphoglyserate kinase (hPGK) promoter. Woodchuckhepatitis virus pre-element (wPRE) and central polypurine tract (cPPT)were used in the vector backbone. The virus production, concentration, andp24 assays were performed as described earlier.12 Titers of the concentratedLVs ranged from 8.5 � 108 to 1.9 � 109 TU/mL. A schematic representa-tion of the LV vector is shown in Figure 1A.

Experimental animals

TG mice were produced by LV transgenesis with the use of the perivitellineinjection method as described.11,13 Briefly, after breeding, fertilized 1-cell–stage oocytes were collected from oviducts of superovulated CD2F1 donorfemales weighing 9 to 12 g. Approximately 100 pL of 109 TU/mLhVEGFD-LV was injected into the perivitelline space of the oocytes. Afterovernight recovery and growth in vitro, the 2-cell–stage embryos weretransferred to pseudopregnant CD2F1 female mice. Pups were weaned andgenotyped at 3 weeks of age. The material for genotype polymerase chainreaction (PCR) was skin attained from earmarking. The genotyping wasperformed by PCR with the use of the following primers: forward primer,5�-TTGCCAGCTCTACCACCAG-3�; reverse primer, 5�-TTCATTGCAA-CAGCCACCAC-3�. The primers were specific for hVEGF-D cDNA.

Animals used in the study were from F0 to F3 generations. TG-negativelittermates (the same genetic and epigenetic background lacking hVEGF-D)were used as controls. All animal studies had the approval of theExperimental Animal Committee of Kuopio University.

Hindlimb ischemia

Hindlimb ischemia model has been described.14 Briefly, mice wereanesthetized with Rompun-Ketalar mixture (10 mg/kg xylazine [Rompun],80 mg/kg ketamine), and ligation of the arteria femoralis superficialis wasperformed. Animals were killed by CO2 inhalation 1, 2, and 3 weeks afterthe operation. The organs were perfused with 1 � PBS via left ventricle.After perfusion, samples from all major tissues were collected for molecu-lar biological and histological analyses. Histologic samples were fixed in4% PFA–15% sucrose for 4 hours, rinsed in 15% sucrose, processed, andembedded in paraffin. Slides (5 �m thick) were cut and routinely stainedwith hematoxylin-eosin (HE).

Copy number and integration site analyses

Mouse genomic DNA was digested with AflII or SacII restriction endonucle-ases for the copy number analysis and for the determination of the numberof integration sites, respectively. Prehybridization of the membranes wasperformed before hybridization with 32P-CTP–labeled wPRE probe. For thecopy number analysis, a standard curve was generated by adding theplasmid amount equal to copy numbers of 1, 5, 10, 20, and 30 into 15 �g ofTG-negative mouse genomic DNA.

Reverse transcriptase–PCR

Expression of hVEGF-D mRNA in tissues of TG mice was analyzed byreal-time reverse transcriptase–PCR (RT-PCR). Total RNAs of differenttissues were extracted by RNeasy kit (QIAGEN, Valencia, CA) accordingto the manufacturer’s instructions. cDNA synthesis was performed with theuse of random hexamer primers and PCR by predesigned hVEGF-Dprimers and probe (Applied Biosystems, Foster City, CA). Normalizationwas performed with the use of mouse �-actin control kit (AppliedBiosystems).

Enzyme-linked immunoabsorbent assays and serum clinicalchemistry

hVEGF-D and mVEGF-A concentrations were analyzed with the use ofQuantikine Immunoassays (Human VEGF-D or Mouse VEGF-A; R&DSystems, Minneapolis, MN). Immunoassays were performed from tissuelysates and from serum samples. Tissues were homogenized in T-PERbuffer (Tissue Protein Extraction Reagent; Pierce Chemical, Rockford, IL),and lysates were prepared according to the manufacturer’s instructions.

Mouse serum samples from TG-negative and hVEGF-D TG mice wereanalyzed with the use of standard clinical chemistry methods at theDepartment of Clinical Chemistry, Kuopio University Central Hospital.

Immunohistochemistry

Paraffin-embedded sections (5 �m thick) were immunostained with thefollowing antibodies: CD31 (platelet endothelial cell adhesion molecule1[PECAM-1], 1:50; BD Biosciences PharMingen, San Diego, CA), CD34(1:20; Hycult Biotechnology, Uden, The Netherlands), LYVE-1 (1:1000,microwave pretreatment in citrate buffer; ReliaTech, Braunschweig, Ger-many), hVEGF-D (1:100, microwave pretreatment in citrate buffer; R&DSystems), VEGFR-2 (1:100; eBiosciences, San Diego, CA), VEGFR-3(1:50; R&D Systems), cytokeratin (CK, 1:50; Affinity BioReagents,Golden, CO), cytokeratin 7 (CK7, 1:50, microwave pr-treatment in citratebuffer; Santa Cruz Biotechnology, Santa Cruz, CA). Immunohistochemicalreactions were developed with the use of ABC Vectastain Elite staining kit(Vector Laboratories, Burlingame, CA) and DAB (Invitrogen Zymed, SanFrancisco, CA).

Light microscopic analysis

Histologic sections were assessed with an Olympus AX-70 microscope(Olympus, Tokyo, Japan) with AnalySIS software (Soft Imaging System,Muenster, Germany). Paraffin slices of all major organs (liver, spleen,pancreas, heart, lungs, salivary glands, testes/ovaries, aorta, limb skeletalmuscles, small intestine, brain, and skin) of TG-positive and TG-negative

Figure 1. Schematic presentation of the third generation lentiviral vector usedin the transgenesis studies and Southern blot image of a copy numberanalysis. (A) CMV indicates cytomegalovirus promoter; �, packaging signal; cPPT,central polypurine tract. The TG cassette contains the human phosphoglyceratekinase (hPGK) promoter, human VEGF-D cDNA truncated from C- and N-termini(hVEGF-D), and woodchuck hepatitis virus PRE element (wPRE). SIN indicates thatthe lentiviral vector is self-inactivating because of the deletion in U3 LTR to form �U3;U5, 5� LTR; RRE, rev-responsive element. (B) Analysis of the 4 hVEGF-D TG foundermice (1, 2, 3, and 4) and 3 F1 mice (2a, 2b, and 2c) from founder number 2. The copynumbers of standards (left half of the image) are indicated (copies/15 �g of mouse tailgenomic DNA). wPRE was used as a probe sequence. Molecular weight marker(MW) between the standard and sample lanes is 2.5 kb. A vertical line has beeninserted to indicate a repositioned gel lane; 1, 1 to 5 copies; 2, 7 to 10 copies; 3, 5 to7 copies; 2a, 5 to 7 copies; 2b, 7 to 10 copies; and 2c, 7 to 10 copies.

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mice were stained with HE, and their histologies were observed for majorphenotypic changes. Hindlimb skeletal muscle (musculus rectus andmusculus caput gastrocnemius) and myocardium slices were stained withCD34 immunohistochemistry and photographed under 20� objective.

Capillary densities (capillaries/myocyte in skeletal muscles, capillaries/mm2 in myocardium) were determined as described.14 Hindlimb skeletalmuscles with ischemic injury were stained with HE. Areas of regenerationand different manifestations of ischemic injury were measured with the useof AnalySIS software (Soft Imaging System). In assessing capillary densityand morphometry, 8 randomly selected fields were analyzed blindly.

Statistical methods

Statistical analyses were performed with GraphPad Prism version 4.00(GraphPad Software, San Diego, CA). Results of the TG-positive andTG-negative groups were analyzed with the use of unpaired, 2-tailed t testwith confidentiality of 95%, considering P values less than .05 asstatistically significant.

Results

Lentiviral transgenesis

We produced hVEGF-D mice with the use of LV transgenesis. Theamount of LV injected into one oocyte was 103 TU. Ninety-onepercent of all injected embryos survived viral microinjection, andon the average 70% of the born founders were TG positive.Southern blot analysis was used to analyze copy numbers of the TGmice (Figure 1B). The copy numbers in all founders were between1 and 10 copies/genome, showing a moderate variation betweenthe mice. Either the same or a lower copy number was detected inF1 offspring. In consecutive generations the copy numbers re-mained similar to the copy numbers of F1 mice.

The number of chromosomal integrations was determined fromF1, F2, and F3 TG mice. Results showed that the provirus wasintegrated into one chromosome in most mice, whereas in one F1

mouse and one F3 mouse, derived from the same founder,2 chromosomal integration sites were found (data not shown).Thus, copy numbers of more than one in most mice are probablydue to concatamerization of the provirus.

In F1 generations the litters were small (2-3 pups), but 90% to100% of the pups were positive for the transgene. As the number ofgeneration advanced, the litter sizes grew, but the number of

TG-positive pups decreased. Nevertheless, the transgene wastransmitted through the germ line from one generation to another.

hVEGF-D mRNA

Expression levels of hVEGF-D mRNA varied between mice, aswell as between tissues in each mouse. However, each RT-PCRrun showed that the same tissues had highest hVEGF-Dexpressions in all generations. The tissues of high expressionwere skeletal muscle, skin, pancreas, and heart (Figure 2A).Most of the other tissues fell into the category of moderateexpression (spleen, lung, kidney, and gonads; Figure 2B),whereas each analysis showed very low or no TG expression inliver and brain (Figure 2B). The expression was easily detect-able in TG mice in all generations.

hVEGF-D protein

Protein expression of hVEGF-D in TG mice is shown in Figure 2Cand D. Concentrations were measured from tissue lysates (Figure2C) and serum (Figure 2D). No hVEGF-D was detected inTG-negative mouse samples. Similar to mRNA levels, the numberof generation of the TG mice did not affect TG protein expressionlevels. The hVEGF-D concentration was slightly lower in the F3

mice than in the F1 mice (Figure 2C). However, the differencebetween the generations was not significant. In serum samples thehVEGF-D protein concentration varied between 20 and 700 pg/mL(Figure 2D).

The results of mVEGF-A enzyme-linked immunoabsorbentassays (ELISAs) showed that hVEGF-D did not affect endogenousexpression of mVEGF-A in TG mice with various copy numbers(data not shown).

Clinical chemistry

Markers of lipid metabolism, liver and kidney function, and tissuedamage were measured from serum of TG-positive and TG-negative mice. Blood for these measurements was collected onkilling. The concentration of total serum cholesterol (Chole),high-density lipoprotein (HDL), low-density lipoprotein (LDL),triglycerides (Trigly), aspartate aminotransferase (ASAT), creati-nine (Crea), lactate dehydrogenase (LD), and creatine kinase (CK)were measured with the use of standard clinical chemistry methods.

Figure 2. Human VEGF-D expression in TG mice.Results of one representative real-time RT-PCR run with3 mice were chosen to show the general pattern of TGexpression in different tissues. F1-1 and F1-2 are mice ofgeneration F1; F3-1, -2 and -3 are TG mice of generationF3. (A) All analyzed tissues are included in the samehistogram. Because the scale is wide, the tissues thatshowed moderate to very low TG expression are shownwith a lower scale in panel B. (C,D) RepresentativeELISA assays for hVEGF-D protein content in TG mice.(C) hVEGF-D protein in tissue lysates of F1 and F3 TGmice. (D) hVEGF-D protein in plasma of 5 F1 mice. Errorbars represent � SD.

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Results of clinical analyses are summarized in Table 1. Nosignificant differences were observed between the groups.

Vascularity

The effect of the transgene on vascular density was measuredfrom intact hindlimb skeletal muscles and myocardium of F0 toF3 mice by CD34 immunohistochemistry. The angiogenic effectwas also determined in mice aged 1 to 2 years to study whetherthe transgene was functional also in aged mice. A representativeimage of CD34 immunostained skeletal muscle is shown inFigure 3A and B. For methodologic comparison, we also show arepresentative image of a serial section immunostained forCD31 in both TG-positive and TG-negative mice (Figure 3C,D,respectively). Results show that constitutively expressed hVEGF-D�N�C is a potent angiogenic factor in mice. The effect of thetransgene on the density of lymphatic capillaries was studiedwith LYVE-1 immunohistochemistry. These stainings showed asimilar number of lymphatic capillaries in TG mice and controlmice. Thus, the hVEGF-D�N�C transgene has no lymphangio-genic effect in the TG mice (Figure 3E,F). Difference in thecapillary count of hindlimb skeletal muscles of nonoperatedTG-negative and TG-positive mice was statistically significant(P � .05; Figure 3G). The number of capillaries in the myocar-dium was also significantly higher in the TG-positive mice thanin the TG-negative littermates (Figure 3H). No major effect wasfound on lymphatic capillary density in skeletal muscles or othertissues. These results suggest that the constitutively expressed

mature form of hVEGF-D is a highly potent angiogenic factorbut not a lymphangiogenic factor in mice.

Regeneration after hindlimb ischemic injury

To study the effect of the transgene expression on mouse musclebiology in pathologic conditions, we induced hindlimb ischemiausing a surgical operation. Signs of tissue damage and regenerationwere studied by morphometrical measurements of the injuredmuscles and by measuring the areas of active regeneration.HE-stained musculus caput gastrocnemius of the TG mice and theTG-negative littermates 7 days after the ischemic injury are shownin Figure 4. When the ischemic area (apoptosis/necrosis, fibrosis,fat atrophy) was measured, 4% to 15% of the total muscle areas inthe hVEGF-D TG mice was damaged, whereas the same range inthe TG-negative mice was 35% to 89%. Signs of active regenera-tion were found in larger areas of ischemic limb muscles in thehVEGF-D TG mice compared with the TG-negative control mice(data not shown).

Spontaneous tumors

On aging the hVEGF-D TG mice showed high frequency ofmalignant tumors (Figure 5). Mice obtained from LV transgenesiswith the same vector backbone and the same promoter but adifferent transgene (human Nrf2) have not developed any tumors(data not shown). In addition, the TG-negative littermates neverdeveloped tumors during the study. Mice with tumors originated

Table 1. Serum clinical analysis of hVEGFD TG mice

Mouse groupChole,mmol/L

HDL,mmol/L

LDL,mmol/L

Trigly,mmol/L

ASAT,U/mL

Crea,�mol/L LD, U/mL CK, U/mL

Controls F1 2.0 � 0.4 1.6 � 0.3 0.13 � 0.04 1.5 � 0.2 1.00 � 0.23 12.3 � 1.5 2.5 � 0.5 23.5 � 5.2

Controls F3 0.9 � 0.1 0.7 � 0.1 0.13 � 0.02 1.2 � 0.1 1.12 � 0.35 12.3 � 1.5 1.6 � 0.3 16.2 � 2.4

Controls all 1.4 � 0.3 1.1 � 0.2 0.13 � 0.02 1.3 � 0.1 1.08 � 0.44 12.3 � 0.1 2.3 � 0.3 21.2 � 3.1

VEGF-D TG F1 1.9 � 0.3 1.5 � 0.4 0.24 � 0.02 1.1 � 0.3 0.61 � 0.14 11.7 � 0.7 1.5 � 0.2 11.9 � 3.1

VEGF-D TG F3 1.3 � 0.3 1.1 � 0.2 0.10 � 0.02 0.8 � 0.2 0.37 � 0.03 9.0 � 1.2 0.7 � 0.1 8.5 � 1.9

VEGF-D TG all 1.5 � 0.2 1.2 � 0.2 0.15 � 0.02 1.3 � 0.2 0.47 � 0.06 10.9 � 0.7 1.2 � 0.07 11.3 � 1.6

Chole indicates cholesterol; HDL, high-density lipoprotein; LDL, low-density lipoprotein; Trigly, triglycerides; ASAT, aspartate aminotransferase; Crea, creatinine; LD,lactate dehydrogenase; and CK, creatine kinase.

A

B

C

D

E

F

G

H

Figure 3. Blood and lymphatic capillary density in TG and control mice as shown by CD34, CD31, and LYVE-1 stainings. Images were taken fromimmunohistochemically stained 5-�m paraffin sections of mouse tissues and analyzed with Olympus Provis AX70 Microscope attached to Olympus ColorView 12 Camera. Thesoftware for imaging and analyses was AnalySIS (Soft Imaging System). (A) Representative figure from a TG mouse showed an increase in capillary density (CD). (B) Incomparison, a normal capillary density was present in control mouse (CD34). (C) Representative figure from a TG mouse showed an increase in capillary density (CD31). (D) Incomparison a normal capillary density was present in control mouse (CD31). (E) Lymphatic vessels were detected only in the interstitium accompanied by venules andarterioles in TG mice (LYVE-1). (F) Control muscles showed a similar pattern and number of lymphatic vessels (LYVE-1). Bars in panels A through F 100 �m.(G) Capillaries/myocyte of nonoperated musculus rectus of the hVEGFD TG mice and the TG-negative controls. The mice were from generations F1, F2, and F3. (H) Capillarydensity in myocardium of the hVEGF-D TG-positive and the TG-negative littermates. The mice were from generations F1, F2, and F3. Error bars represent � SD.

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from 2 different founders. This implies that the integration site isnot involved in the tumor formation. Unexpectedly, half of thetumors were mammary adenocarcinomas, and one of the mice withmammary gland tumor was a male (aged 9 months). In addition,2 lung adenocarcinomas and 2 skin carcinomas were found (Table 2).No lymphomas or other types of lymphatic malignancies were found inthe TG mice.

Characterization of tumors

Most of the mammary gland tumors were poorly differentiated(grade 3) with a solid and comedo growth pattern. One of the micewith tumors was lactating when the tumor was noticed. In one ofthe mice the mammary gland tumor metastasized into the lungs.hVEGF-D was expressed in the surface layer of the ductalcomponent, but not in the solid parts of the tumor. Activeangiogenesis was detected in most tumors. Well-differentiatedmultifocal adenocarcinomas in the lungs, as well as skin tumors(basal cell carcinoma and anaplastic carcinoma) were found insome mice. In the lung tumor, the papillary and stroma of thepapilla stalk were hVEGF-D positive. Anaplastic skin carcinomashowed focal hVEGF-D positivity and intratumoral angiogenesis.A representative panel of the immunostained tumor samples isshown in Figure 6. Detailed characterization of the tumors ispresented in Table 2. All tumors showed detectable amounts ofhVEGF-D mRNA with high or moderate expression levels.

Discussion

The high success rate of the LV transgenesis is most likely due tothe LV perivitelline injection method, compared with the tradi-tional transgenesis method based on plasmid microinjection intothe oocyte pronucleus. In contrast to the perivitelline injection, inthe traditional plasmid microinjection method the oocyte mem-brane must be pierced to bring the plasmid into the pronucleus. Inan alternative LV transgenesis method, the zona pellucida must beremoved, whereas in the LV perivitelline injection technique it isleft in place to cover the oocyte during migration through theoviduct into the uterus and during implantation of the embryo.Thus, the LV perivitelline injection TG method results in asignificantly higher number of implanted embryos and transgenicfounders.11 Another advantage of the perivitelline injection methodover the zona removal is that the fertilized oocytes are transducedat the 1-cell stage. Thus, the transgene is incorporated into each cellof the developing embryo, and mosaicism is usually avoided.

To our knowledge all published studies of LV transgenesis todate were performed only up to the F1 generation. Therefore, wealso wanted to study whether the transgene still was functional inthe TG mice of consecutive generations and in TG mice aged from1 to 2 years. Our results suggest that the hPGK-driven transgeneswere integrated into genomic sites, where they most probably werenot subjected to methylation,15 and that the hPGK promoter can beused to achieve constitutive long-term expression of transgenes.Our results also suggest that LV transgenesis leads to a long-termand effective expression of transgenes in successive generations.

All TG mice with constitutive hVEGF-D expression showednormal growth, were fertile, and appeared generally healthy withno obvious macroscopic phenotype. This implies that constitutivehVEGF-D expression is well tolerated during embryonicdevelopment.

Overexpression of VEGFR-2–specific ligands promotes angio-genesis and lymphatic vessel enlargement but no lymphatic vesselsprouting.16 Therefore, we hypothesized that the hVEGF-D TGmice could have a proangiogenic phenotype. Indeed, increasedblood capillary density was found in skeletal muscles and myocar-dium of the TG-positive mice compared with the TG-negativelittermates. Moreover, after ischemic hindlimb injury, muscleregeneration was faster, and injured areas were significantlysmaller, suggesting that hVEGF-D overexpression improves heal-ing capacity of muscle tissue. These results are consistent with ourprevious findings that intramuscular and intracardiac adenoviral

A B Figure 4. Morphologic characteristics in musculuscaput gastrocnemius 7 days after hindlimb ischemiaoperation. Images were taken from HE-stained 5-�mparaffin sections of mouse tissues and analyzed withOlympus Provis AX70 Microscope attached to OlympusColorView 12 Camera. The software for imaging andanalyses was AnalySIS (Soft Imaging System). (A) In-creased regeneration shown in a hVEGF-D TG mouse onday 7 after hindlimb ischemia operation. Whole areashowed late regeneration features as eosinophilic cyto-plasm and internalization of nuclei. Bar, 200 �m.(B) Control group showed large area of necrosis with paleflocculated cytoplasm (*). Early stage of regeneration isrepresented by basophilic small myocytes with internal-ized nuclei (#). Bar, 200 �m.

Figure 5. Kaplan-Mayer survival curve showing tumor mortality of hVEGF-D TGmice and TG-negative controls. This curve presents all hVEGF-D TG mice thatdied of cancer, regardless of their original background or generation. There were2 high-expressing founders and 1 founder with lower level of expression. First4 points represent mice that died of mammary gland tumor before the age of 1 year.The TG-negative controls had no tumors in any generation during the follow-up.

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hVEGF-D gene transfers increased vascularity and improved bloodflow in transduced tissues.14,17,18 Thus, mature hVEGF-D is primar-ily a proangiogenic growth factor, whether used as a transgene inmice, or applied with the use of local gene therapy in largeranimals, implying that the effects are mostly mediated by VEGFR-2.

Until now, only mice with skin-specific overexpression offull-length hVEGF-D have been generated, whereby the humankeratin 14 (hK14) promoter was used to drive the expression of thetransgene.9 The hK14 promoter directs the TG expression into thebasal cells of the epidermis. These mice showed strong andselective hyperplasia of skin lymphatic vessels but not of skinblood vessels. Furthermore, the lymphatic capillaries in the skinwere dilated and 3-fold larger than those of the wild-type mice.However, in the hK14–hVEGF-D study,9 a different form ofhVEGF-D was used than in the present study. The processed formof hVEGF-D–hVEGF-D�N�C (formed by proteolytical processionof N- and C-terminal ends of the hVEGF-D prepropeptide) almostexclusively binds to VEGFR-2, leading to angiogenesis.6,7,17 Theunprocessed or full-length hVEGF-D, which was used as thetransgene in the hK14–hVEGF-D study,9 equally stimulates phos-phorylation of both VEGFR-2 and VEGFR-3.6

In the present study we have produced TG mice by using a LVthat, when integrated into the host cell genome, directly producesthe hVEGF-D�N�C and does not increase the amount of full-length,unprocessed hVEGF-D. In addition, the difference in the VEGFRbinding capabilities between human and mouse further explains theangiogenic phenotype of the TG mice. In mouse, both proteolyti-cally processed and unprocessed mVEGF-D almost solely bind tomVEGFR-3, leading to enhanced lymphangiogenesis.6,7,10 In addi-

tion, it has been shown that the hVEGF-D�N�C in mouse binds tothe angiogenic mVEGFR-2 and not the lymphangiogenic mVEGFR-3.10,17 These findings most probably explain the different pheno-types regarding the blood and lymphatic vasculature in the2 studies, as well as offer a logical explanation for the absence oflymphangiogenesis in the present study.

The role of VEGF-D in vivo has also been studied by creatingVEGF-D knockout (KO) mice.19 These mice showed a normalphenotype, including normal lymphatic vasculature, and the re-searchers thus concluded that either the mVEGF-D does not have amajor role in lymphangiogenesis during the embryonic develop-ment or that it is compensated by VEGF-C or some other, yetunknown, VEGFR-3–activating agents.19 Because mVEGF-D iscapable of binding only to mVEGFR-3, it is not surprising thatthese KO mice did not have an angiogenic phenotype.

In addition to increased capillary density and improved muscleregeneration after injury, the phenotype of hVEGF-D TG mice alsoincluded a tendency to form tumors. In addition, half of the tumorswere primary mammary gland adenocarcinomas. In the presentstudy, we observed hVEGF-D expression in the well-differentiatedductal part of the mammary adenocarcinomas, in lung papillaryadenocarcinomas, and in skin anaplastic carcinomas, suggesting apathogenetic role of hVEGF-D in tumorigenesis. However, bloodand lymphatic vessel–derived malignancies, such as hemangioma,lymphangioma, angiosarcoma, and lymphangiosarcoma, were notfound. There was no expression of CD31, CD34, or LYVE-1outside the vasculature. Nevertheless, we observed intratumoralangiogenesis, but not lymphangiogenesis, in the samples of mam-mary adenocarcinomas and skin anaplastic carcinomas. The mouse

Table 2. Histopathologic and immunohistochemical characterization of tumors

Organ and tumor type Histology Immunohistochemistry MetastasesGeneration, sex,

comment

Mammary gland

Adenocarcinoma Solid, comedo, trabecular growth

pattern, single acinar/ductal

structures, necrosis, grade 3

hVEGF-D positive in ductal part (surface);

CD31, CD34, LYVE-1 negative in tumor

cells

F1, male, left cervical

mammary gland

Adenocarcinoma Solid, comedo growth pattern,

necrosis

Mammary gland preserved at the

edge, grade 3

hVEGF-D positive in ductal part; CK7

positive; CD31, CD34, LYVE-1 negative

in tumor cells; increased number of

vessels in tumor; angiogenic features

F3, female, left inguinal

mammary gland,

lactating, skin

ulcerated

Adenocarcinoma Tubular, nestal, papillar, and

trabecular growth pattern;

necrosis

Mammary gland preserved at the

edge, grade 3

hVEGF-D positive in ductal part (surface);

CK7 positive; CD31, CD34, LYVE-1

negative in tumor cells; increased

number of vessels in tumor; angiogenic

features

Lung metastases F1, female, right

inguinal mammary

gland

Adenocarcinoma Tubular growth pattern, in situ

component,mammary gland

largely preserved, grade 1

hVEGF-D positive in ductal part (surface);

CD31, CD34, LYVE-1 negative in tumor

cells; increased amount of vessels in

tumor; angiogenic features

F1, female, right

abdominal

mammary gland

Lung

Adenocarcinoma Multifocal, foci of

bronchioloalveolar

proliferation, grade 1

hVEGF-D negative F1, female

Papillary adenocarcinoma Multifocal, papillary growth

pattern, grade 1

hVEGF-D positive in stroma of papilla stalk;

CD31, CD34, LYVE-1 negative in tumor

cells

F1, male

Skin

Basal cell carcinoma Predominantly solid growth of

round basal cells, necrosis

hVEGF-D negative; CD31, CD34, LYVE-1

negative in tumor cells

F3, female

Anaplastic carcinoma Solid infiltrative growth of spindle

and round cells, necrosis,

infiltrating soft tissues (muscle,

bone)

hVEGF-D positive in some tumor cells;

CD31, CD34, LYVE-1 negative in tumor

cells; increased amount of vessels in

tumor; angiogenesis features

F1, male

HUMAN VEGF-D TRANSGENIC MICE 4473BLOOD, 30 APRIL 2009 � VOLUME 113, NUMBER 18

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A B C D

E F G H

I J K L

M N O P

Q R S T

U V W X

Figure 6. Representative histology of tumors in hVEGF-D TG mice. Images were taken from HE-stained or immunohistochemically stained paraffin sections of mouse tissues andanalyzed with Olympus ProvisAX70 Microscope attached to Olympus ColorView 12 Camera. The software for imaging and analyses wasAnalySIS (Soft Imaging System). (A) Mammaryadenocarcinoma with a predominantly solid growth pattern showed focal ducts. Bar, 200 �m. The objective lens used was UPlanApo 4�/0.16 �/ (Olympus). (B) Higher magnification ofthe solid part of mammary carcinoma shown in panelAwith atypical cells and a few mitotic figures. Bar, 50 �m. The objective lens used was UPlanApo 20�/0.70 (Olympus). (C) Mammaryadenocarcinoma with a solid growth pattern displaying only single remnants of ducts. Bar, 200 �m. The objective lens used was UPlanApo 4�/0.16 �/ (Olympus). (D) Highermagnification of the solid part of mammary carcinoma shown in panel C with atypical cells and a few mitotic figures. Bar, 50 �m. The objective lens used was UPlanApo 20�/0.70(Olympus). (E) Mammary adenocarcinoma showing a tubular growth pattern. Bar. 200 �m.The objective lens used was UPlanApo 4�/0.16 �/ (Olympus). (F) Higher magnification of thecarcinoma shown in panel E showing both tubular and solid areas. Bar, 50 �m. The objective lens used was UPlanApo 20�/0.70 (Olympus). (G) Well-differentiated mammaryadenocarcinoma showing a tubular growth pattern. Bar, 200 �m. The objective lens used was UPlanApo 4�/0.16 �/ (Olympus). (H) Higher magnification of the carcinoma shown inpanel G. Bar, 50 �m. (I) Immunostaining for hVEGF-D was positive in the surface of ductular structures. Picture from carcinoma is shown in panels E and F. Bar, 100 �m.The objective lensused was UPlanApo 10�/0.4 Ph1 �/0.17 (Olympus). (J) Solid parts of tumor shown in panels E and F showed CK7 immunopositivity. Bar, 200 �m. The objective lens used was UPlanApo4�/0.16 �/ (Olympus). (K) Increased number of vessels with angiogenic features in mammary adenocarcinoma shown in panels C and D. Bar, 200 �m. The objective lens used wasUPlanApo 4�/0.16 �/ (Olympus). (L) Lung with metastases of mammary adenocarcinoma. Original tumor shown in panels E and F. Bar, 50 �m. The objective lens used was UPlanApo20�/0.70 (Olympus). (M) Higher magnification of the lung metastasis shown in panel L. Note dilated ducts. Bar, 200 �m. The objective lens used was UPlanApo 4�/0.16 �/ (Olympus).(N) Lung papillary adenocarcinoma. Bar, 50 �m. (O) Higher magnification of the adenocarcinoma papillae in tumor shown in panel N. Bar, 50 �m. The objective lens used was UPlanApo20�/0.70 (Olympus). (P) Positive immunostaining for hVEGF-D in papilla stalk. Note positivity also in vessels at the border of the tumor. Bar, 100 �m. The objective lens used wasUPlanApo 10�/0.4 Ph1 �/0.17 (Olympus). (Q) Multifocal foci of bronchioloalveolar proliferation in lung adenocarcinoma. Bar, 200 �m. The objective lens used was UPlanApo 4�/0.16�/ (Olympus). (R) Higher magnification of the bronchioloalveolar proliferation in adenocarcinoma shown in panel R. Bar, 50 �m. The objective lens used was UPlanApo 20�/0.70(Olympus). (S) Solid growth in skin basal cell carcinoma. Note necrosis. Bar, 200 �m.The objective lens used was UPlanApo 4�/0.16 �/ (Olympus). (T) Higher magnification of the basalcell carcinoma. Note mitosis. Bar, 50 �m. The objective lens used was UPlanApo 20�/0.70 (Olympus). (U) Skin anaplastic carcinoma consists of spindle cells. Bar, 200 �m. The objectivelens used was UPlanApo 4�/0.16 �/ (Olympus). (V) Higher magnification of the anaplastic carcinoma. Note atypia. Bar, 50 �m. The objective lens used UPlanApo 20�/0.70 (Olympus).(W) Some tumor cells were positive for hVEGF-D immunostaining in an anaplastic carcinoma. Bar, 100 �m. The objective lens used was UPlanApo 10�/0.4 Ph1 �/0.17 (Olympus).(X) Increased number of vessels with angiogenic features in an anaplastic carcinoma shown in panels U and V. Bar, 100 �m. The objective lens used was UPlanApo 10�/0.4 Ph1 �/0.17(Olympus).

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strain used in this study (CD2F1) is not known for spontaneoustumor formation. However, LVs could be connected to tumorigen-esis. Therefore, we compared the hVEGFD TG mice with otherLV-produced TG mice, namely hPGK-GFP and hPGK-hNrf2. Inthese TG mice, no malignancies or early deaths have been recorded(data not shown).

In line with our findings, tumor angiogenesis has been shown tobe induced by hypoxia, estrogens, VEGF-A, and VEGF-D inprimary breast carcinomas.20,21 Because myoepithelia plays animportant role in mammary gland tumorigenesis, one possiblemechanistic explanation could be the overexpression of VEGF-Din these cells. In previous studies, the role of VEGF-D was seenmainly in the context of lymph node metastasis and intratumorallymphangiogenesis.20,22 Nevertheless, in breast cancer cultures, anautocrine role of hVEGF-D and other members of the VEGFfamily has been implicated.22,23

In conclusion, the hVEGF-D TG mice showed a significantlyincreased angiogenesis in skeletal muscles and myocardium, andless muscle damage after hindlimb ischemic injury compared withthe TG-negative controls. However, the incidence of tumor forma-tion was significantly increased in the TG mice, with a preferencefor mammary gland adenocarcinoma formation.

Acknowledgments

We thank the technical staff of the Department of Biotechnologyand Molecular Medicine for their expert contribution to the study.

This work was supported by the Academy of Finland, FinnishFunding Agency for Technology and Innovation, Kuopio Univer-sity Foundation, European Union (Lymphangiogenomics: ISLH-2007-00284/Ha-7), and Leducq Foundation/Fondation Leducq (06CVD 04).

Authorship

Contribution: A.-M.K. designed, coordinated and performed re-search, analyzed data, and wrote the paper; A.K. performedhistologic analyses, analyzed data, contributed to the animal work,and prepared the images; J.H. performed transgeneses; I.K. ana-lyzed all tumor data and reported the results of the analyzedtumors; S.E.H. performed each hindlimb ischemia surgical opera-tion; A.S. performed most of the additional immunohistochemistry;M.H.D. and E.H. contributed to molecular biology analyses; H.P.set up analysis for expression levels; P.I.M. originally cloned theLV backbone vector; M.P.T. performed ELISA assays; and S.Y.-H.contributed to conception and design of the research, criticallyrevised the manuscript for important intellectual content, andsupervised the research.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.

Correspondence: Seppo Yla-Herttuala, University of Kuopio,A.I.Virtanen Institute, Department of Biotechnology and Molecu-lar Medicine, Neulaniementie 2, 70210 Kuopio, Finland; e-mail:seppo.ylaherttuala@uku.fi.

References

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VEGF-D is the strongest angiogenic and lym-phangiogenic effector among VEGFs deliveredinto skeletal muscle via adenoviruses. Circ Res.2003;92:1098-1106.

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online December 10, 2008 originally publisheddoi:10.1182/blood-2008-07-171108

2009 113: 4468-4475  

Petri Turunen and Seppo Ylä-HerttualaMikkoStefanska, Marike Hinke Dijkstra, Hanna Purhonen, Eveliina Hämäläinen, Petri Ilmari Mäkinen,

Anna-Mari Kärkkäinen, Antti Kotimaa, Jenni Huusko, Ivana Kholova, Suvi Elina Heinonen, Anna and increased susceptibility to tumor formationblood capillary density, improved postischemic muscle regeneration, Vascular endothelial growth factor-D transgenic mice show enhanced 

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