Dopamine Regulates Angiogenesis in Normal Dermal Wound Tissues

Cutaneous wound healing is a normal physiological process and comprises different phases. Among these phases, angiogenesis or new blood vessel formation in wound tissue plays an important role. Skin is richly supplied by sympathetic nerves and evidences indicate the significant role of the sympathetic nervous system in cutaneous wound healing. Dopamine (DA) is an important catecholamine neurotransmitter released by the sympathetic nerve endings and recent studies have demonstrated the potent anti-angiogenic action of DA, which is mediated through its D2 DA receptors. We therefore postulate that this endogenous catecholamine neurotransmitter may have a role in the neovascularization of dermal wound tissues and subsequently in the process of wound healing. In the present study, the therapeutic efficacy of D2 DA receptor antagonist has been investigated for faster wound healing in a murine model of full thickness dermal wound. Our results indicate that treatment with specific D2 DA receptor antagonist significantly expedites the process of full thickness normal dermal wound healing in mice by inducing angiogenesis in wound tissues. The underlined mechanisms have been attributed to the up-regulation of homeobox transcription factor HoxD3 and its target α5β1 integrin, which play a pivotal role in wound angiogenesis. Since D2 DA receptor antagonists are already in clinical use for other disorders, these results have significant translational value from the bench to the bedside for efficient wound management along with other conventional treatment modalities

Anti-Transforming Growth Factor ß Antibody Treatment Rescues Bone Loss and Prevents Breast Cancer Metastasis to Bone

Breast cancer often metastasizes to bone causing osteolytic bone resorption which releases active TGFβ. Because TGFβ favors progression of breast cancer metastasis to bone, we hypothesized that treatment using anti-TGFβ antibody may reduce tumor burden and rescue tumor-associated bone loss in metastatic breast cancer. In this study we have tested the efficacy of an anti-TGFβ antibody 1D11 preventing breast cancer bone metastasis. We have used two preclinical breast cancer bone metastasis models, in which either human breast cancer cells or murine mammary tumor cells were injected in host mice via left cardiac ventricle. Using several in vivo, in vitro and ex vivo assays, we have demonstrated that anti-TGFβ antibody treatment have significantly reduced tumor burden in the bone along with a statistically significant threefold reduction in osteolytic lesion number and tenfold reduction in osteolytic lesion area. A decrease in osteoclast numbers (p = 0.027) in vivo and osteoclastogenesis ex vivo were also observed. Most importantly, in tumor-bearing mice, anti-TGFβ treatment resulted in a twofold increase in bone volume (p<0.01). In addition, treatment with anti-TGFβ antibody increased the mineral-to-collagen ratio in vivo, a reflection of improved tissue level properties. Moreover, anti-TGFβ antibody directly increased mineralized matrix formation in calverial osteoblast (p = 0.005), suggesting a direct beneficial role of anti-TGFβ antibody treatment on osteoblasts. Data presented here demonstrate that anti-TGFβ treatment may offer a novel therapeutic option for tumor-induced bone disease and has the dual potential for simultaneously decreasing tumor burden and rescue bone loss in breast cancer to bone metastases. This approach of intervention has the potential to reduce skeletal related events (SREs) in breast cancer survivors

Doxorubicin-Mediated Bone Loss in Breast Cancer Bone Metastases Is Driven by an Interplay between Oxidative Stress and Induction of TGFβ

<div><p>Breast cancer patients, who are already at increased risk of developing bone metastases and osteolytic bone damage, are often treated with doxorubicin. Unfortunately, doxorubicin has been reported to induce damage to bone. Moreover, we have previously reported that doxorubicin treatment increases circulating levels of TGFβ in murine pre-clinical models. TGFβ has been implicated in promoting osteolytic bone damage, a consequence of increased osteoclast-mediated resorption and suppression of osteoblast differentiation. Therefore, we hypothesized that in a preclinical breast cancer bone metastasis model, administration of doxorubicin would accelerate bone loss in a TGFβ-mediated manner. Administration of doxorubicin to 4T1 tumor-bearing mice produced an eightfold increase in osteolytic lesion areas compared untreated tumor-bearing mice (<i>P</i> = 0.002) and an almost 50% decrease in trabecular bone volume expressed in BV/TV (<i>P</i> = 0.0005), both of which were rescued by anti-TGFβ antibody (1D11). Doxorubicin, which is a known inducer of oxidative stress, decreased osteoblast survival and differentiation, which was rescued by N-acetyl cysteine (NAC). Furthermore, doxorubicin treatment decreased Cu-ZnSOD (SOD1) expression and enzyme activity <i>in vitro</i>, and treatment with anti-TGFβ antibody was able to rescue both. In conclusion, a combination therapy using doxorubicin and anti-TGFβ antibody might be beneficial for preventing therapy-related bone loss in cancer patients.</p></div

Anti-TGFβ antibody inhibits doxorubicin-mediated increase in osteoclast formation.

<p>Mononuclear cells from spleen (A) and bone marrow (B) from C57BL/6 were isolated and cultured for 15 days in the presence of MCSF and RANKL until mature osteoclasts are formed and scored using TRAP staining as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078043#s4" target="_blank">Materials and Methods</a>. (C) Osteoblast-mediated osteoclast formation was also done using a co-culture system as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078043#s4" target="_blank">Materials and Methods</a>. Student T-test was performed and P<0.05 was considered significant.</p

Anti-TGFβ antibody rescues doxorubicin mediated bone loss in breast cancer bone metastasis.

<p>MDA-MB-231 cells (1×10⁶) were inoculated via left cardiac ventricle of four-week old female athymic nude mice and treated with PBS, doxorubicin (5 mg/Kg, once weekly for 4 weeks, i.p.), 1D11 (10 mg/Kg, three times per weekly for 4 weeks) and a combination of doxorubicin and 1D11 for three weeks. (A) Representative microCT images of mice tibiae from each treatment group (B) Quantification of trabecular bone volume (BV/TV), (C) Histology of mice tibia from each group revealed trabecular bone loss upon doxorubicin treatment, both of which were rescued upon anti-TGFβ antibody treatment. Quantification of (D) average trabecular numbers and (E) average trabecular thickness. Representative microCT images from tumor-bearing mice in which (F) 4T1 cells (10⁵) were inoculated either via intracardiac route or (H) in the #4 mammary fat pad of four-week old female Balb/c mice which received either PBS, doxorubicin (5 mg/Kg, once weekly for 4 weeks, i.p.), 1D11 (10 mg/Kg, three times per weekly for 4 weeks) or a combination of doxorubicin and 1D11 for three weeks. Quantification of microCT images show significant loss of trabecular bone volume (BV/TV) in both intracardiac (G) and orthotopic (I) models. At least 5 mice were assessed in each group and P<0.05 was considered significant.</p

Anti-TGFβ antibody treatment improves doxorubicin-mediated inhibition of osteoblast differentiation and increases the frequency of osteoblast colony forming units.

<p>Mouse bone marrow cells were flushed and allowed to attach for two days. Bone marrow stromal cells were trypsinized and replated as 1×10⁶ cells per well in six well plates for fibroblast colony forming units (CFU-F) and 2×10⁶ cells for osteoblast colony forming units (CFU-OB). Cells were cultured either using DMEM-F12 media (10% FBS) alone or supplemented with either doxorubicin (0.01 ug/ml), 1D11(25 µg/ml) or a combination of both until fibroblast colonies were formed. CFU-OB were cultured using osteoblast differentiation media (alpha-MEM+10% FBS) containing ascorbic acid and β glycerophosphate with similar concentration of doxorubicin (0.01 µg/ml) and/or 1D11(25 µg/ml). Upon microscopic colony formation, media were aspirated, plates were washed in PBS, fixed with 10% neutral buffered formalin and stained to score (A) Average number of fibroblast colony forming units (CFU-F) per 1×10⁶ bone marrow cells. (B) Average number of osteoblast colony forming units (CFU-OB) per 2×10⁶ bone marrow cells. (C) Ex <i>vivo</i> osteoblast mineralization assay was performed using mouse calverial osteoblasts isolated from 3 days old pups and plated in triplicate. Upon confluence, cells were grown in osteoblast differentiation media containing ascorbic acid and β glycerophosphate, in presence of doxorubicin (0.01 µg/ml), 1D11(25 µg/ml) or a combination until mineralized matrix were formed. Von Kossa staining was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078043#s4" target="_blank">Materials and Methods</a> and mineralization was scored using Metamorph software in each set and compared with media only group. Student T-test was performed to calculate p-values. P>0.05 was considered significant. N = 6 for each group was used in this experiment. (D) RT-PCR for expression of RANKL, OPG, OCN and OPN from MC3T3 cells treated with media alone, doxorubicin (0.01 µg/ml, 20 hours), anti-TGFβ antibody (25 ug/ml) and a combination of doxorubicin and 1D11.</p

Doxorubicin mediates bone loss by elevating oxidative stress.

<p>(A) C400 oxidation show increase in reactive oxygen species (ROS) upon <i>in vitro</i> doxorubicin (0.01 ug/ml, 20 hours) treatment increases oxidative stress in the mouse bone marrow stromal cells, which was decreased by concomitant treatment with 1D11(25 µg/ml). Data represents average percentage of C400 positive cells from triplicate samples. (B) RT-PCR showing a decrease in SOD1 (copper zinc superoxide dismutase 1) and GPx expression was noted in MC3T3 mouse osteoblast cells upon treatment with doxorubicin (0.01 µg/ml, 20 hours), which was returned to normal level by co-treatment with anti-TGFβ antibody 1D11(25 ug/ml). (C) SOD1 expression normalized against GAPDH expression, quantified by Image J. (D) GPx expression normalized against GAPDH expression, quantified by Image J. (E) SOD1 activity was performed using MC3T3 cells as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078043#s4" target="_blank">Materials and Methods</a> section. The inhibition of SOD1 activity was measured by formation of NBT-diformazan from NBT following 20 hours treatment in either serum free alpha-MEM media alone, or supplemented with 0.01 ug/ml doxorubicin, 25 µg/ml anti-TGFβ antibody and a combination of both doxorubicin and anti-TGFβ antibody. A drastic inhibition of SOD1 activity was noted following doxorubicin treatment which was restored by anti-TGFβ antibody. (F) Calvarial osteoblasts from wild type mice (3–4 days old pups) were cultured until confluent and treated with osteoblast differentiation media supplemented with doxorubicin (0.01 µg/ml), N-acetyl cysteine (NAC, 20 mM) treatment, or a combination of both, or media alone until mineralized matrix was formed. Quantification of Von Kossa staining images from at least 3 different fields were done using Metamorph software.</p