Biopolymer Molecular Weight Can Modulate the Wound Healing Efficacy of Multivalent Sonic Hedgehog-Hyaluronic Acid Conjugates.

There is a clinical need for new therapeutics to improve healing of chronic impaired wounds. Thus, we investigated how biopolymer conjugation could be used to improve the wound healing performance of a key growth factor for tissue regeneration: Sonic hedgehog (Shh). We generated two multivalent Shh conjugates (mvShh) using hyaluronic acid with two different MWs, which exhibited equivalent potency and proteolytic protection in vitro. Using db/db diabetic mice, we showed that mvShh made with smaller HyA MW resulted in more rapid and robust neovascularization compared to mvShh made with larger MW HyA. Further, smaller mvShh conjugates resulted in faster wound resolution compared to the unconjugated Shh. This study is the first to show how the wound healing efficacy of multivalent protein-polymer conjugates is sensitive to the polymer MW, and our findings suggest that this parameter could be used to enhance the efficacy of growth factor conjugates.

Multivalent Conjugates of Sonic Hedgehog Accelerate Diabetic Wound Healing.

Despite their preclinical promise, few recombinant growth factors have been fully developed into effective therapies, in part, due to the short interval of therapeutic activity after administration. To address this problem, we developed nanoscale polymer conjugates for multivalent presentation of therapeutic proteins that enhance the activation of targeted cellular responses. As an example of this technology, we conjugated multiple Sonic hedgehog (Shh) proteins onto individual hyaluronic acid biopolymers to generate multivalent protein clusters at defined ratios (i.e., valencies) that yield enhanced Shh pathway activation at equivalent concentrations relative to unconjugated Shh. In this study, we investigated whether these multivalent conjugates (mvShh) could be used to improve the therapeutic function of Shh. We found that a single treatment with mvShh significantly accelerated the closure of full-thickness wounds in diabetic (db/db) mice compared to either an equivalent dose of unconjugated Shh or the vehicle control. Furthermore, we identified specific indicators of wound healing in fibroblasts and endothelial cells (i.e., transcriptional activation and cell migration) that were activated by mvShh in vitro and at concentrations approximately an order of magnitude lower than the unconjugated Shh. Taken together, our findings suggest that mvShh conjugates exhibit greater potency to activate the Shh pathway, and this multivalency advantage improves its therapeutic effect to accelerate wound closure in a diabetic animal model. Our strategy of multivalent protein presentation using nanoscale polymer conjugates has the potential to make a significant impact on the development of protein-based therapies by improving their in vivo performance.

The dual roles of homeobox genes in vascularization and wound healing.

Homeobox genes represent a family of highly conserved transcription factors originally discovered to regulate organ patterning during development. More recently, several homeobox genes were shown to affect processes in adult tissue, including angiogenesis and wound healing. Whereas a subset of members of the Hox-family of homeobox genes activate growth and migration to promote angiogenesis or wound healing, other Hox genes function to restore or maintain quiescent, differentiated tissue function. Pathological tissue remodeling is linked to differential expression of activating or stabilizing Hox genes and dysregulation of Hox expression can contribute to disease progression. Studies aimed at understanding the role and regulation of Hox genes have provided insight into how these potent morphoregulatory genes can be applied to enhance tissue engineering or limit cancer progression.

Isolation and expansion of endothelial progenitor cells derived from mouse embryonic stem cells.

The unlimited differentiation and proliferation capacity of embryonic stem cells represents a great resource for regenerative medicine. Here, we describe a method for differentiating, isolating, and expanding endothelial cells (ECs) from mouse embryonic stem cells (mESCs). First, mESCs are expanded on a mouse embryonic fibroblast (mEF) feeder layer and partially differentiated into embryoid bodies (EBs) by growing the cells in an ultra-low attachment plate for up to 5 days. The EBs are then differentiated along the endothelial lineage using endothelial growth medium supplemented with 40 ng/mL vascular endothelial growth factor (VEGF). The differentiated endothelial population expresses both Fetal Liver Kinase 1 (Flk-1) and VE-Cadherin on the cell surface which can be further purified using a fluorescence-activated cell sorting (FACS) system and subsequently expanded on 0.1 % gelatin-coated plates. The differentiated cells can be analyzed by real-time PCR and flow cytometry to confirm enrichment of EC-specific genes and proteins.

Temporal changes in Hox gene expression accompany endothelial cell differentiation of embryonic stem cells.

In pluripotent embryonic stem cells (ESCs), expression of the Hox master regulatory transcription factors that play essential roles in organogenesis, angiogenesis, and maintenance of differentiated tissues, is globally suppressed. We investigated whether differentiation of endothelial cells (ECs) from mouse ESCs was accompanied by activation of distinct Hox gene expression profiles. Differentiation was observed within 3 days, as indicated by the appearance of cells expressing specific endothelial marker genes (Flk-1+ /VE-Cadherin+ ). Expression of HoxA3 and HoxD3, which drive adult endothelial cell invasion and angiogenesis, peaked at day 3 and declined thereafter, whereas expression of HoxA5 and HoxD10, which maintain a mature quiescent EC phenotype, was low at day 3, but increased over time. The temporal and reciprocal changes in HoxD3 and HoxA5 expression were accompanied by corresponding changes in expression of established downstream target genes including integrin ß3 and Thrombospondin-2. Our results indicate that differentiation and maturation of ECs derived from cultured ESCs mimic changes in Hox gene expression that accompany maturation of immature angiogenic endothelium into differentiated quiescent endothelium in vivo.

HOXA9 regulates BRCA1 expression to modulate human breast tumor phenotype.

Breast cancer 1, early onset (BRCA1) expression is often reduced in sporadic breast tumors, even in the absence of BRCA1 genetic modifications, but the molecular basis for this is unknown. In this study, we identified homeobox A9 (HOXA9) as a gene frequently downregulated in human breast cancers and tumor cell lines and noted that reduced HOXA9 transcript levels associated with tumor aggression, metastasis, and patient mortality. Experiments revealed that loss of HOXA9 promoted mammary epithelial cell growth and survival and perturbed tissue morphogenesis. Restoring HOXA9 expression repressed growth and survival and inhibited the malignant phenotype of breast cancer cells in culture and in a xenograft mouse model. Molecular studies showed that HOXA9 restricted breast tumor behavior by directly modulating the expression of BRCA1. Indeed, ectopic expression of wild-type BRCA1 phenocopied the tumor suppressor function of HOXA9, and reducing BRCA1 levels or function inhibited the antitumor activity of HOXA9. Consistently, HOXA9 expression correlated with BRCA1 in clinical specimens and with tumor aggression in patients lacking estrogen receptor/progesterone receptor expression in their breast tissue. These findings indicate that HOXA9 restricts breast tumor aggression by modulating expression of the tumor suppressor gene BRCA1, which we believe provides an explanation for the loss of BRCA1 expression in sporadic breast tumors in the absence of BRCA1 genetic modifications.

HOXA3 modulates injury-induced mobilization and recruitment of bone marrow-derived cells.

The regulated recruitment and differentiation of multipotent bone marrow-derived cells (BMDCs) to sites of injury are critical for efficient wound healing. Previously we demonstrated that sustained expression of HOXA3 both accelerated wound healing and promoted angiogenesis in diabetic mice. In this study, we have used green fluorescent protein-positive bone marrow chimeras to investigate the effect of HOXA3 expression on recruitment of BMDCs to wounds. We hypothesized that the enhanced neovascularization induced by HOXA3 is due to enhanced mobilization, recruitment, and/or differentiation of BMDCs. Here we show that diabetic mice treated with HOXA3 displayed a significant increase in both mobilization and recruitment of endothelial progenitor cells compared with control mice. Importantly, we also found that HOXA3-treated mice had significantly fewer inflammatory cells recruited to the wound compared with control mice. Microarray analyses of HOXA3-treated wounds revealed that indeed HOXA3 locally increased expression of genes that selectively promote stem/progenitor cell mobilization and recruitment while also suppressing expression of numerous members of the proinflammatory nuclear factor kappaB pathway, including myeloid differentiation primary response gene 88 and toll-interacting protein. Thus HOXA3 accelerates wound repair by mobilizing endothelial progenitor cells and attenuating the excessive inflammatory response of chronic wounds.

Restoring transcription factor HoxA5 expression inhibits the growth of experimental hemangiomas in the brain.

Hemangiomas are angiogenesis-dependent benign vascular tumors that can rupture and cause intracranial hemorrhages. We previously showed that the transcription factor homeobox A5 (HoxA5), which is absent in activated angiogenic endothelial cells can block angiogenesis. Here, we investigated whether restoring expression of HoxA5 blocks hemangioma growth by transplanting mouse hemangioendothelioma endothelial cells (EOMA) or HoxA5-expressing EOMA cells into the brains of mice. The EOMA cells induced brain hemangiomas characterized by large cystlike spaces lined by thin walls of endothelial cells surrounded by scant smooth muscle cells. When HoxA5-expressing EOMA cells were injected, lesion volumes were reduced between 5- and 20-fold compared with the EOMA control group (p < 0.05). Restoration of HoxA5 was associated with increased thrombospondin-2, which inhibits angiogenesis and reduced hypoxia-inducible factor 1alpha expression. These data suggest that restoring HoxA5 can attenuate experimental brain hemangioma development.

HoxA5 stabilizes adherens junctions via increased Akt1.

Normal vascular development and angiogenesis is regulated by coordinated changes in cell-cell and cell-extracellular matrix (ECM) interactions. The Homeobox (Hox) family of transcription factors coordinately regulate expression of matrix degrading proteinases, integrins and ECM components and profoundly impact vascular remodeling. Whereas HoxA5 is down regulated in active angiogenic endothelial cells (EC), sustained expression of HoxA5 induces TSP-2 and blocks angiogenesis. Since HoxA5 is also lacking in EC in proliferating hemangiomas, we investigated whether restoring expression of HoxA5 could normalize hemangioma cell morphology and/or behavior. Sustained expression of HoxA5 in the murine hemangioma cell line (EOMA) reduced their growth in vivo and promoted branching morphogenesis in 3D BM cultures. Moreover, restoring HoxA5 expression increased the retention of beta-catenin in adherens junctions and reduced permeability. In addition we also show that the HoxA5 mediated increase in stability of adherens junctions requires Akt1 activity and introduction of constitutively active myr-Akt in EOMA cells also increased retention of beta-catenin in adherens junctions. Finally we show that HoxA5 increases Akt1 mRNA, protein expression and further enhances Akt activity via a coordinate down regulation of PTEN. Together these results demonstrate a central role for HoxA5 in coordinating a stable vascular phenotype.

Homeobox D10 induces phenotypic reversion of breast tumor cells in a three-dimensional culture model.

Homeobox (Hox) genes are master regulatory genes that direct organogenesis and maintain differentiated tissue function. We previously reported that HoxD10 helps to maintain a quiescent, differentiated phenotype in endothelial cells by suppressing expression of genes involved in remodeling the extracellular matrix and cell migration. Here we investigated whether HoxD10 could also promote or maintain a differentiated phenotype in epithelial cells. We observed that HoxD10 expression is progressively reduced in epithelial cells as malignancy increases in both breast and endometrial tumors. Retroviral gene transfer to restore expression of HoxD10 in the malignant breast tumor cells MDA-MB-231 significantly impaired migration, and when these cells were cultured in a three-dimensional laminin-rich basement membrane (3DlrBM) model, they formed polarized, acinar structures. This phenotypic reversion was accompanied by decreased alpha3 integrin expression and reduced proliferation. Importantly, expression of HoxD10 in the MDA-MB-231 cells inhibited their ability to form tumors in mouse xenografts. Taken together, our results suggest that HoxD10 has tumor-suppressive functions for mammary epithelial cells.

The homeobox transcription factor Hox D3 promotes integrin alpha5beta1 expression and function during angiogenesis.

Neovascularization promotes wound healing, tumor growth, and arthritis. Endothelial cell migration and survival during neovascularization are regulated by adhesion proteins, including integrin alpha5beta1. Integrin alpha5beta1 is poorly expressed on normal quiescent blood vessels, but its expression is induced on tumor blood vessels and in response to angiogenic factors such as basic fibroblast growth factor, interleukin-8, tumor necrosis factor-alpha, and the angiomatrix protein Del-1. We show here that alpha5beta1 expression, and hence function, during angiogenesis is regulated by the transcription factor Hox D3, a homeobox gene that also controls the expression of endothelial cell integrin alphavbeta3 and urokinase-type plasminogen activator. Hox D3 expression in endothelial cells enhances integrin alpha5 protein and message expression, whereas Hox D3 antisense inhibits its expression. Hox D3 promotes alpha5 expression during angiogenesis in vivo, whereas inhibition of alpha5 expression by Hox D3 antisense suppresses angiogenesis. Hox D3 binds directly to the promoters of the integrin alpha5 and beta3 subunits, inducing subunit expression. As Hox D3, integrin alphavbeta3, and integrin alpha5beta1 are expressed on tumor blood vessels but not on normal quiescent vessels, these studies suggest that Hox D3 coordinately regulates the expression of integrin alpha5beta1 and integrin alphavbeta3 during angiogenesis in vivo. These studies also suggest that Hox D3 inhibition could be a useful approach to inhibit tumor angiogenesis.

HoxD3 expression and collagen synthesis in diabetic fibroblasts.

Diabetic wound healing is characterized by deficiencies in both growth factor and collagen production. We have observed that expression of homeobox D3 (HoxD3), a collagen-inducing transcription factor, and expression of collagen are reduced in an established animal model of diabetic wound repair, the leptin-deficient diabetic (db/db) mouse. We sought to evaluate whether the diminished expression of collagen and HoxD3 would be maintained once fibroblasts were removed from the diabetic wound environment. Fibroblasts were isolated from both wild-type and diabetic animals and expression of HoxD3 and collagen assessed. We found that when removed from the diabetic wound environment, HoxD3 and type I collagen expression are increased in diabetic fibroblasts when compared to wild-type fibroblasts. The increase in type I collagen is not related to increased production or activation of transforming growth factor-beta1. However, when the diabetic fibroblasts are cultured in a 3D collagen matrix, expression of type I collagen and HoxD3 is markedly reduced and reflects the pattern of gene expression observed in the in vivo diabetic wound environment. Thus, although diabetic fibroblasts can regain the capacity to express high levels of collagen and HoxD3 once removed from the diabetic wound environment, culturing cells in the presence of a 3D collagen matrix is sufficient to revert these fibroblasts to their previous nonsynthetic state.

Organized living: from cell surfaces to basement membranes.

Binding of extracellular matrix (ECM) proteins to integrin receptors initiates intracellular signaling events that are essential for the differentiation and survival of epithelial cells. However, the propagation and processing of these signals also depend on the cells acquiring an appropriate three-dimensional morphology and polarity after contact with the ECM. In fact, even if adhesion to the ECM is maintained but subsequent cellular organization and polarity are impaired, epithelial cells fail to fully differentiate and become susceptible to apoptotic stimuli. Studies using three-dimensional tissue culture models with reconstituted basement membranes not only demonstrate the central role of tissue organization for differentiation and survival, but also emphasize how acquiring this organized polarized phenotype can override a number of genetic changes that would otherwise disrupt normal tissue function.

Abnormal expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in brain arteriovenous malformations.

BACKGROUND AND PURPOSE: Excessive degradation of the vascular matrix by matrix metalloproteinases (MMPs) can lead to structural instability of vessels. In this study we examined the expression of MMPs and tissue inhibitors of metalloproteinases (TIMPs) in brain arteriovenous malformations (BAVMs). METHODS: We performed gelatin zymography for MMPs and Western blot for MMP-9, MMP-2, TIMP-1, TIMP-2, TIMP-3, and TIMP-4. MMP-9 expression was localized by immunohistochemistry. RESULTS: We analyzed 37 BAVM specimens and 9 control brain specimens from epilepsy surgery. Thirty-two BAVM patients had embolization treatment before resection. Eighteen BAVM patients had a history of hemorrhage from BAVMs. Neither MMP-2 nor TIMP-2 was detected in BAVMs or control brain specimens. Compared with control brain samples, BAVM samples had higher levels of total MMP-9, active MMP-9, pro-MMP-9, TIMP-1, and TIMP-3. TIMP-4 levels were higher in the control brain than in BAVM specimens. MMP-9 was localized to the endothelial cell/peri-endothelial cell layer and infiltrating neutrophils of BAVMs. BAVMs with venous stenosis >or=50% had higher expression of MMP-9 than BAVMs with venous stenosis <50%. There was no apparent association between total MMP-9, pro-MMP-9, or active MMP-9 levels and (1) feeding artery pressure, (2) pattern of draining vein (exclusively deep venous drainage versus any superficial drainage), and (3) BAVM size. CONCLUSIONS: We found increased levels of MMP-9 and TIMPs in BAVMs. Abnormal balance of MMP-9 and TIMPs may contribute to vascular instability of BAVMs.