Background
Patients with VHL disease are heterozygous for
VHL mutations, and develop tumors when the function of the remaining wild-type
VHL allele is lost via somatic mutation or epigenetic silencing [
1]. VHL tumors, which can occur in several different tissues, are characterized by hypervascularity and a clear cell appearance in histological preparations.
VHL mutations are also frequently observed in sporadic renal cell carcinoma (ccRCC). In addition, specific
VHL missense mutations have been described that do not cause tumors, but result instead in recessive polycythemia, a disease characterized by an overproduction of erythrocytes [
2‐
4].
VHL protein (pVHL) is an essential negative regulator of the hypoxia-inducible factor (HIF), a transcription factor induced by low oxygen tension [
5]. HIF induces a metabolic switch from oxidative phosphorylation to glycolysis, which is essential for cell survival under hypoxic conditions. HIF also promotes angiogenesis and erythropoiesis through induction of cytokines such as vascular endothelial growth factor (VEGF) and erythropoietin (EPO). The active HIF transcription factor is a dimer consisting of an α and a β subunit [
1,
5]. The β unit—known as HIF-1β or ARNT (arylcarbon receptor nuclear translocator)—is ubiquitously and constitutively expressed. In contrast, the HIF-α subunits (HIF-1α, HIF-2α and HIF-3α) are regulated by oxygen tension. Under normoxic conditions, HIF-α is hydroxylated. The hydroxylated form is recognized by an ubiquitin ligase and undergoes ubiquitination, followed by proteasome-mediated degradation. Hydroxylation is oxygen dependent, and is inhibited under hypoxic conditions. Thus, hypoxia leads to stabilization of the HIF-α protein, allowing formation of the dimeric HIF transcription factor and transactivation (or repression) of HIF responsive genes.
pVHL is the substrate recognition component of the multimeric ubiquitin-ligase complex that mediates HIF-α ubiquitination [
1,
5].
VHL gene inactivation therefore leads to normoxic stabilization of HIF-α and inappropriate activation of the HIF transcription factor. The formation of VHL tumors is thought to be driven in large part by genes induced or suppressed by HIF [
5]. However, loss of
HIF-independent functions of
VHL, and mutations of additional tumor suppressor genes, likely also contribute to tumorigenesis [
1,
6,
7]. Recent animal model and cancer genome studies have indicated that
VHL mutations are necessary but insufficient for tumorigenesis [
6,
8‐
10]. Such second and even third hits conceivably can be additional genetic or epigenetic changes within the same cells, or can be within a separate cell population that contributes to the formation of tumor microenvironment. The requirement for additional tumor suppressor gene(s) in ccRCC formation was supported by the construction of
Vhlh (mouse allele of
VHL)
-Bap1 double knockout [
11].
BAP1 gene mutations have been observed in ~10 % of ccRCC samples [
9,
10].
Vhlh-Bap1 double knockout generated clear-cell lesions that resemble carcinoma [
11]. On the other hand, mutations in the cancer stromal cells, including those of the well-known tumor suppressor genes
p53 and
PTEN, have been documented that contribute to cancer progression {reviewed in [
12]}. It is therefore possible that in VHL patients,
VHL inactivation could also occur in the tumor microenvironment (stroma) in addition to the tumor itself.
One of the most frequently observed tumors in VHL patients besides ccRCC is hemangioblastoma, a highly vascularized tumor with extramedullary hematopoiesis that occurs in the central nervous system and the retina [
13]. Hemangioblastomas cause considerable morbidity and mortality despite being benign. Hemangioblastomas are sometimes referred to as vascular tumors; however, biallelic inactivation of
VHL was detected in the stromal compartment of the vascular tumors [
14‐
16], which also have a clear cell appearance. Vascular overgrowth is therefore likely induced by pro-angiogenic cytokines released by these “stromal cells.” In addition, hemangioblastomas frequently contain foci of extramedullary erythropoiesis and the
VHL
−
stromal cells exhibit multipotency that may be of embryonic origin [
17‐
19]. There are no mouse models that recapitulate hemangioblastoma. However, several VHL mouse models develop hemangiomas—an overgrowth of irregularly shaped and leaky blood vessels—in the liver [
20‐
23]. Hemangiomas have been observed in the liver of germline
Vhlh+/- mice [
20] and in mosaic
Vhlh biallelic deletion mice induced by conditional
β-actin-driven
Cre [
21]. These two models contain heterozygous and homozygous, respectively,
Vhlh mutants in most cell types, including hepatocytes and endothelial cells. More interestingly, liver hemangiomas were also observed in
PEPCK-Cre driven
Vhlh knockout, which inactivates
Vhlh in renal proximal tubule cells and in ~20 to 30 % of hepatocytes [
20,
22]. Likely due to early mortality, full-blown hemangiomas were not observed when a more hepatocyte-specific
Cre driver,
Albumin-Cre, was used to inactivate
Vhlh; nonetheless, numerous blood-filled vascular cavities, and foci of increased vascularization within the hepatic parenchyma were observed [
20,
22]. Inactivation of
Vhlh in hepatocytes with
PEPCK-Cre or
Albumin-Cre also led to erythrocytosis—overproduction of erythrocytes—due to increased expression of Epo [
20,
22], although hemangioma-associated extramedullary erythropoiesis—as observed in hemangioblastoma—was not observed. Hif-2α was found to mediate up-regulation of erythropoietin and multiple pro-angiogenic cytokines in these mouse models, and inactivation of
Hif-2α or
Hif-1β/Arnt, but not
Hif-1α, rescued hemangiomas in
PEPCK-Cre or Albumin-Cre driven
Vhlh knockout mice [
22,
24].
Previously we showed that inactivation of
Vhlh in a subpopulation of kidney distal tubules, using the
HOXB7-Cre driver, resulted in
Hif-1α-dependent hyperplastic clear-cell lesions and severe inflammation and fibrosis [
25]. Here, we report that the same
HOXB7-Cre driven
Vhlh conditional knockout mice also developed liver hemangiomas as well as extramedullary erythropoiesis. Interestingly, in contrast to the previous mouse models, we did not detect
Vhlh inactivation in hepatocytes. Instead,
Vhlh inactivation was detected in liver granulocytes in the knockout mice. In support of a myeloid component in the development of hemangiomas in the liver, reconstitution of the knockout mice with wild-type hematopoietic stem cells partially rescued the hemangioma phenotype. Further analysis showed that the granulocyte population contained the
Vhlh deleted allele. In addition, granulocytes (neutrophils) in livers of the
HOXB7-Cre driven
Vhlh knockout mice were found to over-express placental growth factor (PlGF) that has been shown to promote angiogenesis. Thus, this mouse model supports the notion that a bone marrow-derived stromal component with
VHL loss-of-function contributes to the development of the full extent of the VHL disease phenotype.
Methods
Animal protocol and mouse strains
All of the procedures were conducted in accordance with the US Public Health Service Policy on Humane Care and Use of Laboratory Animals. Mice used in these studies were maintained in Boston University Medical Center facility according to protocols approved by the Institutional Animal Care and Use Committee. Mouse strains used were in C57BL/6 background and have been described previously [
25]. For generation of bone marrow chimeras, B6.SJL-
Ptprc
a
Pepc
b
/BoyJ (“B6 CD45.1”) as well as RosaLacZ was purchased from Jackson Laboratories (Bar Harbor, Maine, USA).
Reagents
Phosphate-buffered saline (PBS), Dulbecco’s phosphate-buffered saline (DPBS), Dulbecco’s modified eagle medium (DMEM) and HEPES were obtained from Gibco/Life Technologies (Carlsbad, CA, USA). Fetal bovine serum (FBS) was obtained from Hyclone (Logan, UT, USA). Sterile 0.5 M EDTA stock solution, pH7.5, was obtained from Boston Bioproducts (Ashland, MA, USA). Fluorescence-activated cell sorting (FACS) buffer was prepared as follows: 0.5 % FBS/2 mM EDTA in DPBS. Red blood cell lysis buffer, Fc-block and antibodies for flow cytometry (except anti-CD45 antibody) were obtained from eBioscience (San Diego, CA, USA). Cell strainers (40 μm or 70 μm) were obtained from ThermoFisher Scientific (Waltham, MA, USA).
Histology and immunohistochemistry
Livers were fixed overnight in 10 % neutral buffered formalin and were embedded in paraffin. 4 μm thick paraffin sections were stained with hematoxylin and eosin (H&E) according to standard procedures. For immunohistochemistry, 4 μm thick paraffin sections were dewaxed, and heat mediated antigen retrieval was performed with citrate buffer, pH 6, for 40 min. Endogenous peroxidase was quenched by incubating sections for 15 min in methanol with 0.3 % H2O2 or peroxidase block (Peroxidased 1, Biocare Medical, Concord, CA). Endogenous biotin was blocked with avidin-biotin blocking kit (Vector Laboratories, Burlingame, CA, USA), followed by 30 min blocking with 3 % or 10 % (GFP stain) normal goat serum (Sigma-Aldrich, St. Louis, MO) in PBS. Staining and washing was performed with PBS, 0.05 % Tween 20 (Sigma-Aldrich). Sections were incubated overnight at 4 °C with primary antibody (1:50 rat anti-CD45, clone 30-F11, Molecular Probes/Life Technologies, Carlsbad, CA, USA; 1:100 rabbit anti-mouse Plgf, Origene/Acris Antibodies, Rockville, MD, USA; 1/500 chicken anti-GFP, Abcam) and incubated for 45 min with appropriate biotinylated secondary antibody (Vector Laboratories) at 1/500 (rabbit and chick secondary) or 1/1000 (rat secondary). After washing 3 × 5 min (CD45) or 4 × 15 min [placental growth factor Plgf), GFP], sections were incubated for 45 min with streptavidin-conjugated horseradish peroxidase (Invitrogen/Zymed, Carlsbad, CA, USA) at 1/1000 (CD45 stain) or with ABC Elite Kit (Vector Laboratories; Plgf, GFP stain). Sections were washed again for 3 × 5 min (CD45) or 4 × 15 min (Plgf, GFP) and were incubated for 5–10 min with peroxidase substrate (DAB, Vector Laboratories). Sections were counterstained with hematoxyline and mounted with permount (ThermoFisher Scientific).
Preparation of single cell suspensions from liver
Livers were dissected out taking care to minimize bleeding, and were rinsed with DPBS to wash off excess blood. For wild-type samples, small pieces of liver were dissected out from the left and median lobe (lobe encasing the gallbladder). For knockout samples, liver pieces containing hemangiomas were dissected out. In some cases hemangioma tissue was pooled from two mice to obtain enough material. Next, liver tissue was minced and resuspended in 5–10 ml digestion buffer consisting of ice-cold DMEM with 5 mg/ml (or 800 U/ml) collagenase (trypsin-free Collagenase, CLS-4, Worthington Biochemical Corporation, Lakewood, NY, USA) and 20 mM HEPES. Digestion was performed at 4 °C for 1–1.5 h in 5-ml round bottom polypropylene tubes with overhead rotation. The digest was then diluted 2-fold in FACS buffer, EDTA was added to a final concentration of 2 mM, and the cell solution was strained through a 70-μm strainer. Next, cells were pelleted at 250 xg for 8 min at 4 °C, and resuspended in ice-cold red blood cell lysis buffer (from eBioscience, San Diego, CA, USA). Red blood cell lysis was performed for 3 min on ice. After washing, liver cells were resuspended in FACS buffer and stained as described below.
Flow cytometry and FACS
Staining was carried out in 1.5-ml tubes. For wash steps, cells were pelleted in a tabletop centrifuge (250 xg at 4 °C for 5 min). After treatment with red blood cell lysis buffer (see preparation of single cell suspensions), cells were resuspended in ice-cold FACS buffer and concentration was adjusted to 1 × 106 cells–5 × 106 cells per 100 μl. Cells were incubated with Fc-block (1:100) for 5 min on ice, before adding primary antibodies. After cells were incubated for 20 min on ice with primary antibodies, live/dead stain was performed. For propidium iodide staining, cells were washed once with 1 ml FACS buffer, and resuspended in FACS buffer with 1 μg/ml propidium iodide (1 mg/ml stock solution obtained from Life Technologies). Cells were then transferred to round bottom polypropylene tubes for sorting/analysis (no washing required after propidium iodide step). For staining with Aqua Blue Live/Dead solution (Life Technologies), cells were resuspended in 1 ml FACS buffer with 1:1000 of Aqua Blue stock solution (Life Technologies; stock solution prepared according to instructions of manufacturer) and were incubated for 15 min at 4 °C with overhead rotation (washing after antibody staining and live/dead staining in one step). Subsequently, cells were washed once with 1 ml FACS buffer, and were resuspended in FACS buffer and transferred to round bottom polypropylene tubes for sorting/analysis; cell suspensions were filtered through a 40-μm strainer. For sorting of CD45+ cells, cells were stained with CD45-APC (1:200; Molecular Probes/Life Technologies), followed by propidium iodide stain (Life Technologies). For quantification and sorting of erythrocyte progenitors, cells were triple stained with the following antibodies: CD45-Percp-Cy5 1:200, TER119-APC 1:100 and CD71-PE 1:200; followed by Aqua Blue Live/Dead stain (Life Technologies). For determination of chimerism in peripheral blood, cells were double-stained with CD45.1-PE and CD45.2-APC at 1:100 and dead cells were gated out according to size (FSC vs SSC plots). Cell sorting and analysis of liver samples was performed with the FACS Aria II SORP (Becton-Dickinson) or Beckman-Coulter Moflo.
Livers were dissected out taking care to minimize bleeding, and were rinsed with 20 ml DPBS to rinse off excess blood. Subsequently, homogenization was performed as described above but without collagenase treatment. After passing the cell suspension through a 70-μm strainer, cells were pelleted (250 xg at 4 °C for 8 min) and resuspended in 20 ml DPBS containing 2 % FBS. For cell counting, a small aliquot was stained with an acridine orange and propidium iodide solution (AO/PI solution, Nexcelcom, Lawrence, MA, USA) according to the instructions of the manufacturer, and counted with the cellometer (Nexcelcom). 4.8 × 105 live cells in 300 μl DPBS were then added to 3 ml of MethoCult GF M3434 (Stem Cell Technologies, Vancouver, BC, Canada) and plated out into two 3-cm tissue culture dishes, following instructions of the manufacturer. BFU-E colonies per plate were quantified in a blinded fashion after 7–8 days, and were averaged for each sample (2 plates per sample).
β-galactosidase staining of organs
Chemicals for staining were obtained from ThermoFisher Scientific. Livers and kidneys were fixed in 4 % paraformaldehyde in PBS for ~4 h at room temperature. Organs were then washed (3 × 30 min) with wash buffer (0.1 M NaH2PO4, 0.1 M Na2HPO4, 2 mM MgCl2, 0.01 % deoxycholate, 0.02 % NP-40) and stained overnight at 4 °C with staining buffer (wash buffer with 5 mM ferrocyanide, 5 mM ferricyanide and 1 mg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal). Organs were photographed, post-fixed for 15 min with 4 % paraformaldehyde in PBS, and were embedded in paraffin. 4 μm thick paraffin sections were prepared and counter-stained with Nuclear Fast Red (Vector Laboratories) and analyzed for β-galactosidase staining.
Isolation of genomic DNA and polymerase chain reaction (PCR) for detecting Vhlh deletion
DNA from liver tissue was obtained using the DNAeasy blood and tissue kit (Qiagen, Valencia, CA, USA) according to instructions of the manufacturer. Sorted cells (10,000-500,000) were pelleted in a table top centrifuge (300 xg, 5 min), frozen in <100 μl DPBS with 2 % FBS, and stored at −80 °C. Subsequently, DNA was obtained with the Qiamp Micro DNA kit (Qiagen) following the protocol for DNA isolation from small amounts of blood.
Vhlh primers for detection of the
Vhlh flox allele and wild-type allele have been described before [
22]; sequences are as follows:
Vhlh-wt/flox forward primer (FW1), ctaggcaccgagcttagaggtttgcg;
Vhlh-wt/flox reverse primer (Rev1), ctgacttccactgatgcttgtcacag. PCR products are ~290 bp (
wt allele) and 460 bp (
floxed allele). The site of the forward primer is lost upon recombination; the
Vhlh-wt/flox primers therefore cannot amplify the recombined/deleted
Vhlh allele. Generic primers were used to detect Cre:
FW, atccgaaaagaaaacgttga;
Rev, atccaggttacggatatagt; Cre-PCR product is ~700 bp.
Vhlh deletion primers were designed as follows: the forward primer (FW2) is situated downstream of the second HindIII restriction site, and upstream of the NdeI restriction site within the 5′ untranslated sequence of the murine
Vhlh gene. The reverse primer (Rev2) is situated downstream of the HindIII restriction site within the first intron of the murine
Vhlh gene [
20]. The sequences are as follows:
Vhlh-del forward primer (FW2): ggaaccatctcttctctgatagagc;
Vhlh–del reverse primer (Rev2): gctggttgcttcagacacaatcttg. The
Vhlh del primers flank exon 1 (see Fig.
4c). In the presence of exon 1, the sequence is very long (~4 kb) and is therefore not amplified under stringent PCR conditions (e.g., short extension time). In the presence of Cre, exon 1 is excised, resulting in a much shorter sequence, allowing amplification of the recombined/deleted
Vhlh allele. The
Vhlh-del PCR product is ~ 800 bp. Identity of the product was confirmed by sequencing.
Serum collection and ELISA for erythropoietin
Peripheral blood was collected in heparinized microcapillaries (ThermoFisher Scientific), and was transferred into collection tubes with clotting activator (BD Microtainer SST, Becton Dickinson, Waltham, MA, USA). Blood was incubated for ~5–10 min at room temperature, and centrifuged at full speed in a table-top centrifuge. The serum (corresponding to supernatant) was collected and stored at −80 °C for up to 4 months. Subsequently, erythropoietin ELISA was performed with Quantikine erythropoietin ELISA kit (RD systems Inc, Minneapolis, MN, USA) according to instructions of the manufacturer.
Preparation of cDNA and quantitative PCR
Liver RNA was isolated using Trizol in combination with Purelink RNA Mini kit (Invitrogen/Life Technologies) according to instructions of manufacturer. DNA was digested with on-column DNAse I digestion kit (Invitrogen/Life Technologies) according to instructions of manufacturer. Sorted cells (~20,000) were pelleted, resuspended in Quiazol (Qiagen) and stored at −80 °C. Subsequently, RNA was purified using the miRNAeasy Kit (Qiagen) according to instructions of the manufacturer. After elution, RNA from sorted cells was dried in GenTegra™-RNA tubes (GenTegra, Pleasanton, CA, USA) and was resuspended in ~5 μl of water. Liver RNA (1 μg) or entire RNA obtained from ~20,000 sorted cells was reverse transcribed with AMV First Strand cDNA kit (liver) or Protoscript II first strand synthesis kit (sorted cells; both kits from New England Biolabs, Ipswich, MA, USA) according to instructions of the manufacturer. Real-time PCR was performed with Power SYBR Green Mastermix (Applied Biosystems/Life Technologies) using a StepOne Real time PCR system (Applied Biosystems/Life Technologies). The following primers from the Universal Probe Library (Roche/Life Technologies) were used: 18 s forward primer, gcaattattccccatgaacg; 18 s reverse primer, gggacttaatcaacgcaagc; erythropoietin forward primer, ccctgctgcttttactctcc; erythropoietin reverse primer, gggggagcacagaggact; prolyl-hydroxylase 3 (Phd3) forward primer, tgtctggtacttcgatgctga; reverse primer, agcaagagcagattcagtttttc.
Hoechst staining and bone marrow transplant
Staining with Hoechst 33342 (Life Technologies) was performed as described previously [
26,
27], with the following modification: to increase the yield, Hoechst was titrated so that cells were understained (to achieve a Hoechst negative side population of ~1 %). Subsequently, only the least Hoechst positive cells (comprising 0.2–0.5 % of all cells) were sorted, and 1000 SP were used per recipient; 3 × 10
5 unfractionated bone marrow cells were co-transplanted to improve survival. Recipients were lethally irradiated one day before transplantation with a split dose of 14 gray (2 month old recipients) or 11 gray (4 week old recipients) separated by 2 h. For 2 weeks after the transplant, starting with the day of the transplant, recipients received antibiotics in the drinking water. To examine chimerism using flow cytometry, peripheral blood was collected in heparinized microcapillaries (ThermoFisher Scientific). Blood samples (~100 μl) were then transferred to 1.5 ml tubes containing 100 μl DPBS with 2 mM EDTA. 1 ml of red blood cell lysis buffer was added to samples, and red blood cell lysis was carried out 5 min at room temperature. After adding 10 ml of ice cold FACS-buffer, samples were transferred to 15 ml centrifuge tubes and were centrifuged (8 min, 250 g, 4 °C). Cells were then resuspended in 100 μl FACS-buffer, and stained with antibodies for flow cytometry (see above).
Statistical analysis
Unpaired, two-tailed t-tests were performed; where necessary, Welch’s correction for unequal variances was applied. All analyses were performed using GraphPad Prism Software (La Jolla, CA, USA). For group comparisons, p-values were calculated with GraphPad prism software, and false discovery rate (FDR) was calculated manually using Bonferroni post-test or Benjamini and Hochberg FDR formula.
Discussion
The kidney phenotype in
HOXB7-Cre; Vhlh
fl/fl
knockout mice develops at ~2 months of age {[
25] and data not shown}. In contrast, nascent hemangiomas are already apparent in 75 % of 4-week old mice, and well-developed hemangiomas are present in 90 % of 6-week old mice (Fig.
1d). Furthermore,
Hif-2α inactivation ameliorates the hemangioma phenotype, but has no effect on the kidney phenotype. Thus, the liver phenotype is most likely not the result of
Vhlh inactivation in the kidney lesion. We cannot exclude the possibility that Hif-2α-induced factors emanating from the kidney are required for liver hemangioma formation. However, this explanation is not favored because other kidney-specific
Vhlh knockouts do not develop liver phenotype [
11,
33].
Liver hemangiomas have been described previously in germline
Vhlh
+/-
mice and in mice with
Vhlh inactivation in hepatocytes (
PEPCK-Cre or
Albumin-Cre driven
Vhlh knockout mice) [
20,
22,
23].
PEPCK-driven
Cre is worth noting because it has dual specificity in kidney (proximal tubules) and liver (~20–30 % hepatocytes) [
22]. Here, we show that
HOXB7-Cre driven Vhlh conditional knockout mice also develop hemangiomas in the liver, although unlike previous models, this
Cre driver does not mediate recombination in hepatocytes. We were unable to detect Cre activity in hepatocytes using the
Rosa-LacZ reporter, and we also did not observe steatosis in hepatocytes, a phenotype characteristic of
Vhlh null hepatocytes [
20,
22].
The more hepatocyte-specific
Cre (
Albumin-Cre) driven
Vhlh knockout also generated hemangiomas, albeit more at the microscopic level [
22]. However, despite superficial similarity of the phenotypes, several findings indicate that the mechanism of hemangioma formation is different in hepatocyte-specific
Albumin-Cre driven and
HOXB7-Cre driven
Vhlh knockout mice. In
HOXB7-Cre driven
Vhlh knockout mice, hemangiomas develop with higher penetrance (90 % vs ~40 %) and at a younger age (4–6 weeks versus ~3–8 months) compared to the
Albumin-Cre Vhlh knockout mice [
20]. Furthermore, the hemangiomas in
HOXB7-Cre driven
Vhlh knockouts exhibit extramedullary hematopoiesis that is also observed in human hemangioblastoma but not in liver with hepatocyte
Vhlh knockout (either
Albumin-Cre or
PEPCK-Cre driven).
Another difference between the hepatocyte-specific
Vhlh knockout mice and the
HOXB7-Cre driven
Vhlh knockout mice is the pattern of erythropoiesis. Erythropoietin is over-expressed in the knockout mice with hepatocyte-specific
Vhlh inactivation [
20,
22], as well as in
HOXB7-Cre driven
Vhlh knockout mice. However, while erythropoietin over-expression induced systemic erythrocytosis—characterized by elevated hematocrit—in
PEPCK-Cre and
Albumin-Cre driven
Vhlh knockout mice [
20,
22], erythropoiesis was only increased in
HOXB7-Cre driven
Vhlh knockout mice in specific organs such as liver. There is no increase in hematocrit in
HOXB7-Cre driven
Vhlh knockouts (data not shown). This pattern of focal erythropoiesis resembles the pattern observed in VHL patients, who develop polycythemia only rarely, despite tumor-associated extramedullary erythropoiesis [
34].
By PCR, a stronger signal for the recombined allele was detected in hemangiomas compared to healthy liver tissue, indicating that Vhlh null cells are enriched in hemangiomas. Also, Cre activity was undetectable with the Rosa-LacZ reporter in mice with wild-type Vhlh. Thus, Vhlh inactivation likely occurs in a cell type that is rare in the healthy liver and undergoes expansion or is recruited to the liver when Vhlh is inactivated.
One type of cells that was enriched in hemangiomas is leukocytes. We detected foci of leukocytes (CD45+) adjacent to hemangiomas, and by PCR we detected
Vhlh inactivation in leukocytes isolated from hemangiomas (Fig.
5). We further identified granulocytes/neutrophils as the
Vhlh mutant cells in this model (Figs.
6 and
7). Furthermore, hemangiomas were partially rescued in
HOXB7-Cre; Vhlh
fl/fl
mice reconstituted with wild-type hematopoietic stem cells (Fig.
8). Thus,
Vhlh inactivation in bone marrow-derived leukocytes contributes to hemangioma formation in this mouse model. Interestingly,
Vhlh null leukocytes were not sufficient to induce hemangiomas, since no hemangiomas were observed in the reverse experiment (wild-type mice reconstituted with
HOXB7-Cre; Vhlh
fl/fl
hematopoietic stem cells). Thus, inactivation of
Vhlh in at least one other cell type besides bone marrow-derived cells is required for hemangioma formation. It may be that
HOXB7-Cre driven
Vhlh knockout in kidney tubule cells contributes to systemic elevation of Epo, which in turn is required for hemangioma formation in the liver. However, kidney hyperplasia and tubule defects were rescued by inactivation of
Hif-1α, but not
Hif-2α [
25], whereas other kidney-specific
Vhlh knockout mouse models did not develop liver hemangioma [
11,
33]. The other contributing cell types might include embryonic hematopoietic progenitor cells in the liver that fail to migrate to the bone marrow. However, we did not detect
Vhlh deletion in the erythrocyte progenitor cells, despite increased number of these progenitors. We did, however, detect
Vhlh deletion in liver granulocytes/neutrophils and moderately increased number of Granulocyte/Macrophage/Megakaryocyte/Erythroid (GMME) progenitors cells in the liver (data not shown). This indicates that
Vhlh mutant liver-resident granulocyte progenitors may be critical for liver extramedullary erythropoiesis and hemangioma formation. Interestingly, it has been reported that
VHL haploid insufficiency in neutrophils contributes to their decreased apoptosis, and thus population expansion, in VHL patients. It may be that liver resident granulocytes/neutrophils undergo expansion and promote angiogenesis. We indeed observed increased population of Plgf-expressing neutrophils in knockout mice compared to wild-type. Therefore both bone marrow-derived and liver-resident granulocytes/neutrophils are needed for the full penetrance of liver hemangioma phenotype.
We have not observed the hemangioma phenotype in other organs of the
Vhlh knockout mice described here. This indicates that a unique hepatic microenvironment may be required. However, since the kidney defects observed in the
Hoxb7-Cre; Vhlh
fl/fl
knockout mice include hyper-vascularization and hemorrhage [
25], it is tempting to speculate that the activated neutrophils may also contribute to the full extent of the kidney hyperplastic phenotypes. Future studies should address this intriguing possibility.
The finding that
Vhlh null granulocytes contribute to hemangioma formation is of significance for understanding hemangioblastoma and other highly vascularized VHL tumors. Since VHL patients are heterozygous for
VHL loss-of-function mutations, it is conceivable that inactivation of the remaining wild-type allele could occur in more than one cell type (as indeed it does, since patients are prone to develop tumors in several tissues), including those constituting the stromal compartments. Mutations in the cancer stromal cells have been documented that contribute to cancer progression {reviewed in [
12]}. In particular, neutrophils have been recognized as a major inducer of tumor angiogenesis [
35]. Significantly, recent reports have identified CXCR4 (a known HIF target)-expressing neutrophils as a highly pro-angiogenic subtype of neutrophils [
36]. It is therefore possible that in VHL patients,
VHL inactivation occurs in components of the tumor microenvironment in addition to the tumor itself. Pro-angiogenic
VHL null granulocytes/neutrophils could therefore contribute to the development of the tumor vasculature.
Acknowledgments
We thank Tracy L. Pritchett and Richard Near for assistance with mouse breeding (TLP, RN) and collection of mouse tissues (TLP). We are grateful to Joel Henderson, Associate Professor of Pathology, who helped interpret the pathology of liver hemangioma and fibrotic lesions. We thank Samir Kamat for testing of primers used for quantitative PCR. Furthermore, we thank the excellent core facilities of the Boston University School of Medicine for assistance: Immunohistochemistry Core (paraffin embedding), Analytical Instrumentation Core (autoblood analyzer) and Flow Cytometry Core (cell sorting and assistance with setting up and analyzing multicolor flow cytometry). We also would like to thank members of the Center for Regenerative Medicine (CReM) at Boston University School of Medicine for help with setting up bone marrow transplants assays: George Murphy, Gustavo Mostoslavsky, and Dolly Thomas.