Abnormal vascular endothelial growth factor expression in mesenchymal stem cells from both osteonecrotic and osteoarthritic hips.
Abstract: In osteonecrosis (ON) of the hip, interruption of angiogenesis is a pathological process that may lead to impairment of the nutrient supply, cell death, and the collapse of bone. However, the process of angiogenesis in ON is not well understood. The purpose of this study was to investigate the expression of vascular endothelial growth factor (VEGF) in human mesenchymal stem cells (MSCs) in vitro. Cultured MSCs obtained from the hips of normal, ON, and osteoarthritic (OA) patients all expressed VEGF-A. Furthermore, MSCs from normal stem cells also expressed VEGF-B, but its expression had a tendency to increase in those stem cells from ON and OA patients, while VEGF-C was absent in all of the stem cells. However, VEGF-D expression consistently decreased in MSCs from ON patients, but increased in stem cells from OA donors over that of control cells. In addition, placental growth factor (PGF), which has a similar function as VEGF, was expressed in MSCs, and the levels were similar in MSCs from normal, ON, and OA donors. The results suggest that ON and OA are associated with aberrant VEGF-D expression.
Article Type: Report
Subject: Osteoarthritis (Development and progression)
Osteoarthritis (Research)
Vascular endothelial growth factor (Physiological aspects)
Vascular endothelial growth factor (Measurement)
Vascular endothelial growth factor (Research)
Bones (Necrosis)
Bones (Development and progression)
Bones (Research)
Authors: Mwale, Fackson
Wang, Hongtian
Johnson, Aaron J.
Mont, Michael A.
Antoniou, John
Pub Date: 01/01/2011
Publication: Name: Bulletin of the NYU Hospital for Joint Diseases Publisher: J. Michael Ryan Publishing Co. Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2011 J. Michael Ryan Publishing Co. ISSN: 1936-9719
Issue: Date: Jan, 2011 Source Volume: 69 Source Issue: 1
Topic: Event Code: 310 Science & research
Accession Number: 289216157
Full Text: Osteonecrosis (ON), also known as avascular necrosis, bone infarction, ischemic necrosis, subchondral osteonecrosis, and aseptic necrosis, occurs when there is cellular death of bone due to the interruption of the blood supply. Without a blood supply, eventually, there will be collapse of the architectural bony structure. If it involves the bones of a joint, such as the hip, knee, or shoulder, ON often leads to articular cartilage damage and destruction of the articular surfaces. This event will lead to joint pain and loss of function and is frequently severe enough to require arthroplasty surgery.

Underneath the collapsing bone in ON of the hip, there is typically an attempt at a reparative response. Unfortunately, except under conditions of extremely small lesions, the reparative response fails, as osteoclastic resorption leads to subchondral collapse. Beneath the collapsing segment, there is often a hypervascular area, with the resultant effect similar to a hypertrophic nonunion found in altered fracture healing. At present, it is not known how to definitively repair the ischemic bone, but one potential treatment method would be to differentiate mesenchymal stem cells (MSCs) from adjacent living bone tissue into osteoblasts, and then embed them within a biomatrix to stimulate angiogenesis. This would test the hypothesis that under the appropriate stimulus, these cells, together with the remaining inorganic mineral volume, could produce a matrix that mimics that of native bone. Hopefully, the repaired tissue would have properties resembling those found in healthy and fully functional bone.

MSCs have been shown to undergo enhanced bone formation in a mouse model of a segmental bone defect in terms of expressing osteogenic and angiogenic factors, such as BMP2+VEGF. (1) This model mimics the cellular condensation requirements for embryonic mesenchymal osteoblastogenesis and provides the biochemical environmental factors conducive to bone formation.

Geiger and colleagues have shown that if the blood supply is compromised in fracture healing, (24) application of osteogenic factors alone cannot induce successful bone healing. Angiogenesis is essential for restoring blood flow to the fracture site. Treatment with vascular endothelial growth factor (VEGF) superfamily members, VEGFA, VEGFB, VEGFC, VEGFD, or placental growth factor (PGF) are key requirements for angiogenesis. (2) Human MSCs have been found to express VEGF, suggesting that implantation of human MSCs is a practical means for a source of VEGF production. (3) Autologous MSCs have appropriate differentiation properties, easy accessibility, and proliferative capacity. Because of the potential similarity to the altered healing response found in fracture nonunions that have been successfully treated with autologous MSCs, they could potentially complement ON treatment by secreting angiogenic factors and undergoing osteoblast differentiation. The purpose of this study was to investigate the expression of the angiogenic VEGF superfamily members in MSCs from normal, ON, and osteoarthritis (OA) patients.

Materials and Methods

Source and Preparation of Stem Cells

The three sources of stem cells used in the study were: 1. normal human MSCs obtained from Lonza Group, Ltd. (Basel, Switzerland); 2. MSCs from OA patients obtained from 15 milliliter aspirates drawn from the intramedullary canal of donors undergoing total hip arthroplasty for OA, using a protocol approved by the research ethics committee (REC) of the Jewish General Hospital; and 3. MSCs from ON patients, obtained in a similar manner, from donors undergoing hip arthroplasty, using the same protocol approved by the institutional review board (IRB) of Sinai Hospital of Baltimore.

Bone marrow aspirates were processed as previously described. (4-7) Briefly, each aspirate was diluted 1:1 with Dulbecco's Modified Eagle Medium (DMEM) reagent (Invitrogen[TM], Burlington, Ontario, Canada) and layered over 1:1 with Ficoll (Ficoll-Paque Plus; GE Healthcare Bio-Sciences, Baie-d'Urfe, Quebec City). After centrifugation at 900 x g for 30 minutes, the mononuclear cell layer was removed from the interface, washed with DMEM, and re-suspended in DMEM supplemented with 10% fetal bovine serum (Hyclone, Logan, Utah), 100 units/ml penicillin, 100 [micro]g/ml streptomycin, and 2 mM L-glutamine. The cells were plated in 20 milliliters of media in a 176-[cm.sup.2] culture dish and incubated at 37[degrees] Celsius (C) in a 5% C[O.sub.2] humidified atmosphere. After 72 hours, nonadherent cells were discarded, and the adherent ones were thoroughly washed twice with DMEM. Thereafter, the cells were expanded, as previously described. (5)

Cell Culture

Approximately, one million of third or fourth passage OA and ON donor MSCs were cultured on commercial polystyrene tissue culture dishes (Sarstedt, Inc., Montreal, Quebec, Canada) in DMEM-high glucose with 10% FBS. The media was changed every 2 days for up to 7 days, after which cells were harvested for gene expression studies.

Total RNA Isolation

Total RNA was extracted from MSCs by a modification of the method of Chomcynski and Sacchi, (8) using TRIzol[R] reagent (Invitrogen[TM]). After centrifugation for 15 minutes at 12,000 x g at 4[degrees]C, the aqueous phase was precipitated in one volume of isopropanol, incubated for 20 minutes at room temperature, and centrifuged again for 15 minutes at 12,000 x g at 4[degrees]C. The resulting RNA pellet was air-dried, re-suspended in 40 [micro]l of diethylpyrocarbonate-treated water, and 5 [micro]l was assayed for RNA concentration and purity by measuring [A.sub.260]/[A.sub.280].

Reverse Transcription (RT) and LightCycler[R]

Real-Time Polymerase Chain Reaction (PCR)

Salt-free primers for target genes VEGFA, VEGFB, VEGFC, VEGFD and PGF, as well as for housekeeping gene GAPDH, were generated by Invitrogen[TM]. The sequences of the primers are shown in Table 1. For LightCycler[R] reaction, every 20 [micro]L reaction solution consists of a master mix of the following reaction components: 8 [micro]L Rnase free distilled water, 10 [micro]L SYBR Green mixture (Qiagen), 0.5 [micro]L forward primer (0.25 [micro]M), 0.5 [micro]L reverse primer (0.25 [micro]M), and 1 [micro]L cDNA.

The real time PCR condition included one cycle of denaturation (95[degrees]C for 15 minutes), 45 cycles of amplification and quantification (95[degrees]C for 15 seconds, 58[degrees]C for 15 seconds, and 72[degrees]C for 15 seconds, with a single fluorescence measurement), melting curve (65[degrees] to 95[degrees]C, with a heating rate of 0.1[degrees]C per second and a continuous fluorescence measurement), and finally a cooling step to 40[degrees] C. After real-time PCR, the samples were collected by centrifugation, and the gene size was analyzed on 2.0% agarose gel. The primer sequences used for PCR shown in Table 1 were chosen because they are specific for human RNA, and they amplify a single product. GAPDH primers have been described in one of our earlier articles. (9)

Calculation of Relative Units of Gene Transcription

The crossing points (CPs) were determined by LightCycler[R] software, version 3.3 (Roche Diagnostics, Basel, Switzerland) and were measured at constant fluorescence level. Every sample was run in duplicate. The relative units of real-time PCR were determined by the following equation:

Relative units = [2.sup.[DELTA]CP target]/[2.sup.[DELTA]CP reference]

Regarding statistical analysis, all experiments were performed in triplicates, and statistical differences between the treated and the controls were analyzed by Statview (SAS Institute, Inc., Cary, North Carolina). Results were considered statistically significant at p < 0.05. All results were the average of three samples [+ or -] standard deviation.

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Results

After culturing the cells for 24 hours, MSCs from normal, ON, and OA donors had the typical appearance of stem cells, with no overt differences between the groups (Fig.1). Normal donors showed strong VEGF-A message (Fig. 2, lane 1). VEGF-A message from ON and OA MSCs were also prominent, although they had a tendency to decrease (Fig. 2, lanes 2 and 3). In view of these results, in which VEGF-A, being the most important member of the VEGF proteins, appeared in all of the stem cells and with the understanding that VEGF-A can stimulate vasculogenesis and angiogenesis, we decided to analyze VEGF B, C, and D, as well as PGF, a growth factor with similar functions as VEGF. MSCs from normal stem cells also expressed VEGF-B; however, in contrast to VEGF-A, its expression had a tendency to increase in stem cells from ON and OA patients (Fig.3).

Since VEGF-C was previously reported to be active in angiogenesis, lymphangiogenesis, endothelial cell growth, survival, and permeability of blood vessels, it appeared possible that MSCs express VEGF-C. However, VEGF-C was not detected in any of the stem cells (data not shown). The expression of VEGF-D was markedly reduced in normal stem cells (Fig. 4, lane 1). Its expression was strongly down-regulated in MSCs from ON but strongly up-regulated in stem cells from OA donors (Fig. 4, lanes 2 and 3). PGF was expressed in MSCs, and the levels were similar in MSCS from normal, ON, and OA donors (Fig. 5).

Discussion

MSCs have the ability to recruit and participate in angiogenesis and de novo arteriogenesis, and VEGF plays a central role in the observed host-derived angiogenic response. Previously, it was proposed that ex vivo expanded autologous MSCs may serve as cell therapy to promote therapeutic angiogenesis. (10) In this and our previous studies, (4,6,7,9,11,12) we used RT-PCR analyses of MSCs, which permitted expression analyses of the interrelationships of the VEGF family members VEGF-A, VEGF-B, VEGF-C, and VEGF-D, as well as PDF genes, that have usually been studied individually concerning their participation in stem cell-mediated angiogenesis in ON and OA donor cells.

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We observed that MSCs from normal, ON, and OA patients were characterized by a distinct signature profile of VEGF gene expression. Thus, MSCs from control patients are characterized by the expression of VEGF-A, VEGF-B, PGF, VEGF-D, and the absence of VEGF-C. This suggests that angiogenesis involving MSCs does not require VEGFC. There is also an absence of or low level of expression of type X collagen, better known as a protein expressed by terminally hypertrophic chondrocytes. (7,12,13)

MSCs from ON patients were characterized by the expression of VEGF-A, VEGF-B, PGF, and decreased expression of VEGF-D for reasons that are unclear. The decreased expression of VEGF-D suggests that angiogenesis in MSCs may not require VEFG-D.

MSCs from OA patients were characterized by a reduced expression of VEGF-A and increased expressions of VEGF-B and VEGF-D. The increased expression of VEGF-B and VEGF-D in these MSCS raises questions as to whether this is related to type X collagen expression. In these cells, it is noteworthy that aggrecan and type X collagen are constitutively expressed. (5-7,12,13) Previously, we have shown that the onset of hypertrophy in the growth plate was accompanied by the strongest but transient expression of VEGF as well as aggrecan. (14)

Various studies have proposed using various VEGF factors to complement osteogenic factors for the treatment of ON in animal models. (15-23) A recent report described the potential use of genetically engineered bone marrow stem cells carrying genes for VEGF and bone morphogenetic protein-6 (BMP-6) (to induce osteogenesis) for the treatment of pre-collapse ON. (16) Another report described the down-regulation of VEGF proteins and gene expression in rabbits with steroid-induced ON. (17) In another report, the expression of VEGF was assessed in six specimens from late stage ON of the femoral head. (18) These investigators found that osteoblasts from the reactive interface exhibited increased VEGF expression, which the investigators postulated might be a secondary phenomenon in an attempt to stimulate ingrowth of a reparative blood supply. They also found that osteoblasts derived from OA femoral heads exhibited down-regulation of VEGF after 24 hours of co-incubation with glucocorticoids. Other studies have assessesed the role of VEGF proteins in various animal models and clinical studies of ON. (19-23)

Conclusion

Our data further define the complex changes and interrelationships in the VEGF family gene expression in stem cells from ON and OA donors that may occur in the course of vascular invasion and cell death. This investigation draws attention to these VEGF molecules and their relationships to the physiological and pathological events that are part of angiogenesis.

Disclosure Statement

None of the authors have a financial or proprietary interest in the subject matter or materials discussed, including, but not limited to, employment, consultancies, stock ownership, honoraria, and paid expert testimony.

References

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Fackson Mwale, Ph.D., Hongtian Wang, Ph.D., and John Antoniou, M.D., Ph.D., are from the Lady Davis Institute for Medical Research and Department of Surgery, SMBD-Jewish General Hospital, McGill University, Montreal (QC), Canada. Aaron J. Johnson, M.D., and Michael A. Mont, M.D., are from Rubin Institute for Advanced Orthopedics, Center for Joint Preservation and Reconstruction, Sinai Hospital of Baltimore, Baltimore, Maryland.

Correspondence: Fackson Mwale, Ph.D., Lady Davis Institute for Medical Research and Department of Surgery, SMBD-Jewish General Hospital, 3755 Cote Ste-Catherine Road, Montreal, Quebec H3T 1E2, Canada; fmwale@ldi.jgh.mcgill.ca.
Table 1 Primers for Human VEGFA, B, C, D, PGF and GAPDH

                                                       PCR
                                                       Product
Gene    Sequence                                       Size

VEGFA   Forward:   GGGCAGAATCATCACGAAGT    (100-119)   211
        Reverse:   TGGTGATGTTGGACTCCTCA    (221-310)
VEGFB   Forward:   CCCTTGACTGTGGAGCTCAT    (163-172)   203
        Reverse:   CACTGGCTGTGTTCTTCCAG    (346-365)
VEGFC   Forward:   CACTTGCTGGGCTTCTTCTC    (4-23)      173
        Reverse:   TGCTCCTCCAGATCTTTGCT    (157-176)
VEGFD   Forward:   TGTAAGTGCTTGCCAACAGC    (565-574)   163
        Reverse:   GTGGATTTTCCTCCTGCAAA    (708-727)
PGF     Forward:   GTTCAGCCCATCCTGTGTCT    (216-235)   163
        Reverse:   AACGTGCTGAGAGAACGTCA    (359-378)
GAPDH   Forward:   TGAAGGTCGGAGTCAACGGAT   (11-31)     181
        Reverse:   TTCTCAGCCTTGACGGTGCCA   (171-191)

PCR, polymerase chain reaction; VEGF, vascular endothelial growth
factor; PGF, placental growth factor; GAPDH, glycerol-3-phosphate
dehydrogenase.
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