Heparin-binding Growth Factors Research Articles

M402, a heparan sulfate mimetic and novel candidate for the treatment of pancreatic cancer.

4056 Background: Recent advances in pancreatic cancer research implicate the involvement of several heparin-binding growth factors (such as HGF, HB-EGF, PDGF, hedgehogs, and TGFs) that control tumor-stroma interactions. We have rationally designed a heparan sulfate mimetic, M402, which has been previously shown to affect tumor progression and metastasis through disruption of multiple pathways. We hypothesized that M402 could modulate tumor-stroma interactions and enhance the efficacy of gemcitabine, and evaluated its efficacy in two preclinical models. Methods: A genetically engineered mouse model (GEMM; KrasLSLG12D p53LSLR172H) featuring spontaneous pancreatic tumor formation and metastasis assessed M402’s effect on tumorigenesis and metastasis. The orthotopic Capan-2 model in nude mice evaluated the effect of M402 on desmoplasia, a fibrotic response that hinders effective delivery of chemotherapeutics, via inhibition of sonic hedgehog (SHH) signaling in fibroblasts and stellate cells. In both models, M402 was studied as monotherapy and with gemcitabine. Results: In the GEMM, M402 significantly prolonged survival in combination with gemcitabine while each monotherapy showed modest efficacy. M402, alone and in combination, also reduced metastases and local invasion and inhibited epithelial-to-mesenchymal transition. In the Capan-2 model, gemcitabine was increasingly less effective as desmoplasia progressed over time. The addition of M402 to gemcitabine increased its efficacy with respect to primary tumor burden. Metastasis, invasion, and surrounding fibrotic lesions appeared particularly impacted by the combination treatment. M402 was also effective as monotherapy with dose-dependency, which correlated with reduced SHH signaling. Conclusions: M402 can modulate tumor-stroma interactions involved in the metastatic and desmoplastic pathways in two pancreatic cancer models supporting the translation of these findings into a clinical study. A first-in-human study is planned that will evaluate the safety, pharmacokinetics, efficacy, and biomarker profiles of escalating M402 doses in combination with gemcitabine in patients with metastatic pancreatic cancer.

Abstract 1524: M402, a novel heparan sulfate mimetic, inhibits pancreatic tumor growth and desmoplasia potentially via sonic hedgehog signaling in an orthotopic mouse model

Abstract One of the hallmarks of pancreatic ductal adenocarcinoma (PDAC) is extensive desmoplasia in the surrounding tumor microenvironment that prevents delivery of chemotherapeutics (such as gemcitabine). Dysregulation of sonic hedgehog (SHH, a heparin-binding morphogen), has been implicated in pancreatic cancer tumorigenesis. We have rationally designed a heparan sulfate mimetic, M402, that has previously been shown in animal studies to reduce metastatic seeding through disruption of several key heparin-binding growth factors (including FGF2, VEGF, SDF-1α and P-Selectin). We hypothesized that M402, alone or in combination with gemcitabine, could potentially modulate tumor-stroma interactions in an orthotopic pancreatic cancer model (Capan-2 cell line) which secretes SHH and displays a desmoplastic response in vivo. Capan-2 human adenocarcinoma cells (∼1x106 cells) were injected into the pancreata of immunodeficient mice (Nu/Nu CD-1). M402 (10-40 mg/kg/day) or saline treatment commenced on Day 4 or 32. Gemcitabine (30-60 mg/kg, twice weekly, IP) treatment commenced between Weeks 3-6. At the termination of each study (Week 8), gross anatomical observations were made of the primary pancreatic tumor and metastatic lesions in the surrounding tissues including the spleen, intestines and liver. Gemcitabine was increasingly less effective when started at later time points, but still reduced the primary tumor weight by 60-70% (at 30-45 mg/kg) when treatment was started at week 5. While the addition of M402 to gemcitabine showed some additive effect on primary tumor burden, metastasis, invasion, and surrounding fibrotic lesions appeared particularly impacted by the combination treatment. M402 was also effective as monotherapy showing a dose-dependent reduction in primary tumor weight and fibrotic lesions. Immunohistochemical and qPCR analyses showed reduced fibrosis and SHH signaling with M402 and gemcitabine combination treatment. These results demonstrate that M402 can modulate tumor-stroma interactions involved in the desmoplastic response in a murine model of pancreatic cancer and provide a rationale for the clinical investigation of M402 as a potential anti-desmoplastic agent in pancreatic cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1524. doi:1538-7445.AM2012-1524

Age-related Changes in Rat Myocardium Involve Altered Capacities of Glycosaminoglycans to Potentiate Growth Factor Functions and Heparan Sulfate-altered Sulfation

Glycosaminoglycans (GAGs) are essential components of the extracellular matrix, the natural environment from which cell behavior is regulated by a number or tissue homeostasis guarantors including growth factors. Because most heparin-binding growth factor activities are regulated by GAGs, structural and functional alterations of these polysaccharides may consequently affect the integrity of tissues during critical physiological and pathological processes. Here, we investigated whether the aging process can induce changes in the myocardial GAG composition in rats and whether these changes can affect the activities of particular heparin-binding growth factors known to sustain cardiac tissue integrity. Our results showed an age-dependent increase of GAG levels in the left ventricle. Biochemical and immunohistological studies pointed out heparan sulfates (HS) as the GAG species that increased with age. ELISA-based competition assays showed altered capacities of the aged myocardial GAGs to bind FGF-1, FGF-2, and VEGF but not HB EGF. Mitogenic assays in cultured cells showed an age-dependent decrease of the elderly GAG capacities to potentiate FGF-2 whereas the potentiating effect on VEGF(165) was increased, as confirmed by augmented angiogenic cell proliferation in Matrigel plugs. Moreover, HS disaccharide analysis showed considerably altered 6-O-sulfation with modest changes in N- and 2-O-sulfations. Together, these findings suggest a physiological significance of HS structural and functional alterations during aging. This can be associated with an age-dependent decline of the extracellular matrix capacity to efficiently modulate not only the activity of resident or therapeutic growth factors but also the homing of resident or therapeutic cells.

The heparin binding growth factors midkine and pleiotrophin regulate the antinociceptive effects of morphine through α2-adrenergic independent mechanisms

Genetic deletion of pleiotrophin (PTN) impairs spinal nociceptive transmission suggesting that this heparin binding growth factor could play roles in acute pain processing. Despite the high functional redundancy between PTN and midkine (MK), the only other member of this family of growth factors, we now demonstrate that genetic inactivation of MK does not alter acute nociceptive transmission since pain responses of female MK genetically deficient (MK−/−) and wild type (WT+/+) mice were found to be similar in the hot-plate and tail-immersion tests. It has also been shown that morphine administration significantly regulates MK levels within the brain, suggesting that MK could play a role in morphine-induced antinociceptive effects. To test this hypothesis, we have now studied morphine-induced antinociceptive effects in female MK−/− and WT+/+ mice. We did not find differences among genotypes using different doses of morphine (2.5, 5 and 10mg/kg) in the hot-plate test. In contrast, we found that morphine significantly delayed pain responses in MK−/− mice compared to WT+/+ mice in the tail-immersion test. In confirmation of previous results from our group, we also found significantly enhanced morphine-induced antinociceptive effects in PTN−/− mice in the tail-immersion test. In addition, we now demonstrate that enhanced morphine analgesic effects in PTN−/− and MK−/− mice are not caused by a different contribution of descending noradrenergic inhibitory pathways since the α2-adrenergic antagonist yohimbine failed to alter morphine-induced analgesia in all genotypes. The data demonstrate that MK is an endogenous modulator of morphine antinociceptive effects, identify significant differences between PTN and MK in the control of pain processing at the spinal level, and support the hypothesis that inhibitors of the PTN/MK signaling pathway could potentiate opioid analgesia which may be relevant in opioid-refractory pain cases.

Novel Experimental and Clinical Therapeutic Uses of Low-Molecular-Weight Heparin/Protamine Microparticles

Low-molecular-weight heparin/protamine microparticles (LMW-H/P MPs) were produced as a carrier for heparin-binding growth factors (GFs) and for various adhesive cells. A mixture of low-molecular-weight heparin (MW: approximately 5000 Da, 6.4 mg/mL) and protamine (MW: approximately 3000 Da, 10 mg/mL) at a ratio of 7:3 (vol:vol) yields a dispersion of microparticles (0.5–3 µm in diameter). LMW-H/P MPs immobilize, control the release and protect the activity of GFs. LMW-H/P MPs can also bind to cell surfaces, causing these cells to interact with the LMW-H/P MPs, inducing cells/MPs-aggregate formation and substantially promoting cellular viability. Furthermore, LMW-H/P MPs can efficiently bind to tissue culture plates and retain the binding of important GFs, such as fibroblast growth factor (FGF)-2. The LMW-H/P MPs-coated matrix with various GFs or cytokines may provide novel biomaterials that can control cellular activity such as growth and differentiation. Thus, LMW-H/P MPs are an excellent carrier for GFs and various cells and are an efficient coating matrix for cell cultures.

Preservation of FGF-2 bioactivity using heparin-based nanoparticles, and their delivery from electrospun chitosan fibers

Here we present a novel matrix-mimetic nanoassembly based on polysaccharides. Chitosan electrospun fiber networks are decorated with heparin-containing polyelectrolyte complex nanoparticles (PCNs) that present basic fibroblast growth factor (FGF-2), both stably adsorbed to the surfaces and released into solution. These FGF-2/PCN complexes can be released from the fibers with zero-order kinetics over a period of 30days. Further modification of fibers with a single bilayer of polyelectrolyte multilayer (PEM) composed of N,N,N-trimethyl chitosan and heparin completely prevent release, and the FGF-2/PCN complexes are retained on the fibers for the duration of the release experiment (30days). We also compare the mitogenic activity of these FGF-2/PCN complexes delivered in two different states: adsorbed to a surface and dissolved in solution. FGF-2/PCN complexes exhibit mitogenic activity with respect to ovine bone marrow-derived mesenchymal stem cells, even after being preconditioned by incubating for 27days at 37°C in solution. However, when the FGF-2/PCN complexes are adsorbed to chitosan and coated with PEMs, the mitogenic activity of the FGF-2 steadily decreases with increasing preconditioning time. This work demonstrates a new system for stabilizing and controlling the delivery of heparin-binding growth factors, using polysaccharide-based matrix-mimetic nanomaterials. This work also contributes to our understanding of the preferred mode of growth factor delivery from porous scaffolds.

Inter-connecting pores of chitosan scaffold with basic fibroblast growth factor modulate biological activity on human mesenchymal stem cells

► Chitosan (CS) solutions were ultrasonicated to modulate molecular weight of chitosan. ► Interconnecting micropores of scaffolds were observed on the ultrasonicated time. ► Using a LBL method, we prepared heparin immobilized CS scaffolds as depot for bFGF. ► From releasing test, we observed sustained release of bFGF from CS scaffolds. ► The defined pores and bFGF on CS scaffold induced the higher in vitro activity. In this study, we developed bio-active molecules immobilized chitosan scaffolds with controlled pore architectures for enhanced viability of human mesenchymal stem cells (hMSCs). The decreasing in molecular weight of chitosan by ultrasonication of chitosan solution was effective in the formation of porous chitosan scaffolds, resulting in an increase of inter-connecting micropores (∼10 μm) between macropores. Using a layer-by-layer method, we then prepared heparin-coated chitosan scaffolds as depots for basic fibroblast growth factors (bFGF). Enzyme-linked immunosorbent assays confirmed that heparin-coated chitosan scaffolds could bind higher amount of bFGF (24.21 ng/mg) compared to 2.53 ng/mg of non-coated scaffold. Moreover, we were able to manipulate the release profiles of bFGF from the scaffolds for 7 days. In vitro studies showed that chitosan scaffolds induced the improved viability and proliferation of hMSCs through their synergetic effects of the inter-connecting micropores and the sustained released of bFGF. Our results suggest that bFGF immobilized chitosan scaffolds with controlled inter-connecting pores, in combination with other heparin-binding growth factors, have potential implants for controlling biological functions in regenerative medicine.

A KrasG12D-driven genetic mouse model of pancreatic cancer requires glypican-1 for efficient proliferation and angiogenesis.

Pancreatic ductal adenocarcinomas (PDACs) exhibit multiple molecular alterations and overexpress heparin binding growth factors (HBGFs) and glypican-1 (GPC1), a heparan sulfate proteoglycan that promotes efficient signaling by HBGFs. It is not known, however, whether GPC1 plays a role in genetic mouse models of PDAC. Therefore, we generated a GPC1 null mouse that combines pancreas-specific Cre-mediated activation of oncogenic Kras (KrasG12D) with deletion of a conditional INK4A/Arf allele (Pdx1-Cre;LSL-KrasG12D;INK4A/Arflox/lox;GPC1-/- mice). By comparison with Pdx1-Cre;LSL-KrasG12D;INK4A/Arflox/lox mice that were wild-type for GPC1, the Pdx1-Cre;LSL-KrasG12D;INK4A/Arflox/lox;GPC1-/- mice exhibited attenuated pancreatic tumor growth and invasiveness, decreased cancer cell proliferation and mitogen activated protein kinase (MAPK) activation. These mice also exhibited suppressed angiogenesis in conjunction with decreased expression of mRNAs encoding several pro-angiogenic factors and molecules, including vascular endothelial growth factor-A (VEGF-A), SRY-box containing gene (SOX17), chemokine C-X3-C motif ligand 1 (CX3CL1), and integrin β3 (ITGB3). Moreover, pancreatic cancer cells isolated from the tumors of GPC1-/- mice were not as invasive in response to fibroblast growth factor-2 (FGF-2) as cancer cells isolated from wild-type mice, and formed smaller tumors that exhibited an attenuated metastatic potential. Similarly, vascular endothelial growth factor-A (VEGF-A) and FGF-2 did not enhance the migration of hepatic endothelial cells and immortalized murine embryonic fibroblasts isolated from GPC1 null mice. These data demonstrate in an oncogenic Kras-driven genetic mouse model of PDAC that tumor growth, angiogenesis and invasion are enhanced by GPC1, and suggest that suppression of GPC1 may be an important component of therapeutic strategies in PDAC.

Heparan Sulfate Proteoglycans Mediate the Angiogenic Activity of the Vascular Endothelial Growth Factor Receptor-2 Agonist Gremlin

Heparan sulfate proteoglycans (HSPGs) modulate the interaction of proangiogenic heparin-binding vascular endothelial growth factors (VEGFs) with signaling VEGF receptor-2 (VEGFR2) and neuropilin coreceptors in endothelial cells (ECs). The bone morphogenic protein antagonist gremlin is a proangiogenic ligand of VEGFR2, distinct from canonical VEGFs. Here we investigated the role of HSPGs in VEGFR2 interaction, signaling, and proangiogenic capacity of gremlin in ECs. Surface plasmon resonance demonstrated that gremlin binds heparin and heparan sulfate, but not other glycosaminoglycans, via N-, 2-O, and 6-O-sulfated groups of the polysaccharide. Accordingly, gremlin binds HSPGs of the EC surface and extracellular matrix. Gremlin/HSPG interaction is prevented by free heparin and heparan sulfate digestion or undersulfation following EC treatment with heparinase II or sodium chlorate. However, at variance with canonical heparin-binding VEGFs, gremlin does not interact with neuropilin-1 coreceptor. On the other hand, HSPGs mediate VEGFR2 engagement and autophosphorylation, extracellular signaling-regulated kinase(1/2) and p38 mitogen-activated protein kinase activation, and consequent proangiogenic responses of ECs to gremlin. On this basis, we evaluated the gremlin-antagonist activity of a panel of chemically sulfated derivatives of the Escherichia coli K5 polysaccharide. The results demonstrate that the highly N,O-sulfated derivative K5-N,OS(H) binds gremlin with high potency, thus inhibiting VEGFR2 interaction and angiogenic activity in vitro and in vivo. HSPGs act as functional gremlin coreceptors in ECs, affecting its productive interaction with VEGFR2 and angiogenic activity. This has allowed the identification of the biotechnological K5-N,OS(H) as a novel angiostatic gremlin antagonist.

Abstract 2811: GPC1 promotes tumor growth and angiogenesis in a KrasG12D- Driven mouse model of pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive malignancy that is highly therapy-resistant and is the fourth leading cause of cancer-related deaths. The poor prognosis is due, in part, to late diagnosis in conjunction with a high frequency of activating Kras mutations, a loss of the tumor suppressor genes p16INK4A/p14ARF, as well as an over-abundance of heparin-binding growth factors (HBGFs) and their co-receptors. Glypican-1 (GPC1), a co-receptor required for efficient signaling by heparin-binding growth factors, is overexpressed in PDAC and plays a critical role in cell growth, metastasis, and angiogenesis of human and mouse cancer cells. While it is known that the loss of GPC1 results in decreased tumor growth and metastasis in orthotopic mouse models, GPC1's role in pancreatic tumor initiation, development, and progression is unknown. Accordingly, we generated a GPC1 null mouse that combines pancreas-specific Cre-mediated activation of oncogenic Kras (KrasG12D) and deletion of a conditional Ink4a/Arf tumor suppressor allele. Loss of GPC1 resulted in attenuated pancreatic tumor growth and invasiveness, as well as decreased angiogenesis, cancer cell proliferation, and mitogen activated protein kinase (MAPK) activation. These differences were first observed in 30 day old mice and were still highly significant in 65 day old mice. Moreover, pancreatic cancer cells isolated from the tumors of GPC1 null mice were not as invasive in response to fibroblast growth factor 2 (FGF2) as cancer cells isolated from wild-type mice. These data demonstrate in a genetic mouse model of PDAC that GPC1 does not contribute to pancreatic cancer initiation, but that even in the presence of two highly prevalent molecular alterations (oncogenic Kras and loss of p16) endogenous GPC1 contributes to PDAC progression by facilitating tumor growth, angiogenesis, and invasion. Our findings support the concept that GPC1 may be a therapeutic target in PDAC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2811. doi:10.1158/1538-7445.AM2011-2811

Abstract 3113: M402, a novel heparan sulfate mimetic, modulates the desmoplastic response in an orthotopic pancreatic cancer model in mice

Abstract Pancreatic ductal adenocarcinoma (PDAC), currently the fourth leading cause of cancer-related deaths in the US, is one of the most lethal cancers due in part to its resistance to or poor delivery of existing chemotherapeutics (such as gemcitabine). One of the hallmarks of this deadly disease is extensive desmoplasia in the surrounding tumor microenvironment. Recent advances in pancreatic cancer research implicate the involvement of several heparin-binding growth factors that control tumor-stroma interactions (including sonic hedgehog (shh), HB-EGF, TGFs, PDGF and HGF). We have rationally designed a heparan sulfate mimetic, M402, that has previously been shown in animal studies to reduce metastatic seeding through disruption of multiple heparin-binding growth factor-mediated pathways. We hypothesized that M402, alone or in combination with gemcitabine, could potentially modulate tumor-stroma interactions in an orthotopic pancreatic cancer model (Capan-2 cell line) which displays moderate desmoplasia in vivo. Capan-2 human adenocarcinoma cells (∼1×106 cells) were injected into the pancreases of immunodeficient mice (Nu/Nu CD-1, female, 8 weeks old). M402 (40 mg/kg/day) or saline treatment commenced on day 4 (via osmotic pump). Gemcitabine (30-60 mg/kg, twice weekly, IP) treatment commenced between weeks 3-6. At the termination of each study (week 8), gross anatomical observations were made of the primary pancreatic tumor and metastatic lesions in the surrounding tissues including the spleen, intestines and liver. The primary pancreatic tumor was weighed and further processed for immunohistochemistry and mRNA expression of fibrotic matrix markers (fibronectin and COLI) and shh signaling (shh and Gli1). Gemcitabine was increasingly less effective when started at later time points, but still reduced the primary tumor weight by 60-70% (30-45 mg/kg) when treatment was started at week 5. M402 was also effective as monotherapy. The addition of M402 to gemcitabine showed only marginally more activity on primary tumor burden; however, metastasis, invasion, and surrounding fibrotic lesions appeared more impacted by the combination treatment than either agent alone. Immunohistochemical and qPCR analyses showed reduced shh and Gli1 with M402 and gemcitabine combination treatment. These results show that M402 can modulate tumor-stroma interactions involved in the desmoplastic response in a murine model of pancreatic cancer. These results provide a rationale for the clinical investigation of M402 as a potential anti-desmoplastic agent in pancreatic cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3113. doi:10.1158/1538-7445.AM2011-3113

Abstract 1563: M402, a heparan sulfate mimetic, inhibits taxane mediated mobilization of endothelial progenitor cells and their homing to tumors in a mouse model

Abstract Treatment with certain anti-cancer agents, particularly taxanes and sunitinib, can lead to mobilization of pro-angiogenic factors and an acute mobilization of Endothelial Progenitor Cells (EPCs), which home to the viable tumor rim where they can enhance tumor vascularization. This phenomenon has been linked to rapid tumor regrowth following chemotherapy or treatment with specific angiogenesis inhibitors and may thus diminish the long-term efficacy of the treatment. EPCs are mobilized in response to circulating growth factors and chemokines (VEGFR, FGF, G-CSF, IL-6, SDF1α, etc.) that are induced by the drug or the progressing tumor. Many of these factors contain heparin binding domains for their anchorage to proteoglycans on cell surfaces or the extracellular matrix. We tested a novel heparan sulfate mimetic, M402, for its ability to inhibit EPC mobilization as well as EPC homing to the tumor as a result of interference with heparin-binding cytokines, chemokines and growth factors. In normal BALB/c mice, G-CSF and Docetaxel induced a significant increase in EPC within 8-24 h of dosing as compared to the saline control, while cisplatin or gemcitabine treatment did not show this effect. The docetaxel-induced EPC mobilization into the blood could be inhibited significantly by M402, similarly to an anti-VEGFR2 antibody (DC101). Analysis of the bone marrow microenvironment indicated that M402 does not inhibit the generation of EPCs but rather prevents the mobilization of EPCs from the bone marrow, likely through disruption of the SDF-1α/CXCR4 axis. M402 alone did not influence EPC mobilization in normal mice. However, in mice bearing orthotopic 4T1 breast carcinoma or Capan-2 pancreatic tumors, treatment with M402 normalized circulating EPCs otherwise mobilized by tumor-secreted factors, particularly G-CSF. Furthermore, M402 inhibited homing of EGFP labeled EPCs into SDF-1a loaded matrigel plugs or 4T1 tumors. The effect on EPCs correlated with decreased tumor angiogenesis as assessed by immuno-histochemistry and microCT. Furthermore, we observed synergistic anti-tumor activity of M402 with docetaxel in the 4T1 model. In conclusion, the experiments show that M402 could inhibit docetaxel or tumor-mediated EPC mobilization by trapping the EPCs in the bone marrow and inhibiting their recruitment into the tumor microenvironment. The data provide a scientific rationale for further studying the observed synergy of M402 with taxanes in cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1563. doi:10.1158/1538-7445.AM2011-1563

Glypican-3 is expressed in rhabdomyosarcomas but not adult spindle cell and pleomorphic sarcomas

AimsGlypican-3 (GPC3) is a membrane-bound heparan sulphate proteoglycan. Glypicans are predominantly expressed during development in cell- and tissue-specific manners and act as co-receptors for many heparin-binding growth factors, playing important...

Role of Hyaluronan-Mediated CD44 Signaling in Head and Neck Squamous Cell Carcinoma Progression and Chemoresistance

Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy that may involve the oral cavity, pharynx, larynx, and paranasal sinuses. The mechanisms of tumor progression underlying the clinical behavior of HNSCC remain unclear. CD44 comprises a family of transmembrane receptors that can give rise to multiple CD44 variant isoforms. Hyaluronan (HA), a major extracellular matrix component is the primary ligand for CD44 receptors. HA and CD44 signaling play an important role in HNSCC progression. Several CD44 variant isoforms (including v3-, v6-, and v10-containing isoforms) are associated with advanced disease, possibly through unique growth factor interactions with binding domains in the inserted variant regions of the cytoplasmic domain of CD44. In HNSCC, HA mediates the formation of a complex including CD44 and the epidermal growth factor receptor (EGFR) which is overexpressed in a large proportion of HNSCCs. Downstream effectors under EGFR regulation are activated, promoting promote cell growth and tumor survival. The leukemia-associated Rho-guanine nucleotide exchange factor (LARG) also associates with CD44 and EGFR to promote several Ras and RhoA pathway effectors, leading to cell migration, growth, and tumor survival. The secretion of matrix metalloproteinases, necessary for tumor cell invasion, is also regulated by these HA/CD44-mediated pathways. Finally, EGFR-mediated pathways play major roles in the HA/CD44 promotion of chemoresistance in HNSCC. Understanding HA/CD44-mediated signaling pathways may lead to improved treatment of HNSCC.

Mechanism of anti-angiogenic property of gold nanoparticles: role of nanoparticle size and surface charge

Discovering therapeutic inorganic nanoparticles (NPs) is evolving as an important area of research in the emerging field of nanomedicine. Recently, we reported the anti-angiogenic property of gold nanoparticles (GNPs): It inhibits the function of pro-angiogenic heparin-binding growth factors (HB-GFs), such as vascular endothelial growth factor 165 (VEGF165) and basic fibroblast growth factor (bFGF), etc. However, the mechanism through which GNPs imparts such an effect remains to be investigated. Using GNPs of different sizes and surface charges, we demonstrate here that a naked GNP surface is required and core size plays an important role to inhibit the function of HB-GFs and subsequent intracellular signaling events. We also demonstrate that the inhibitory effect of GNPs is due to the change in HB-GFs conformation/configuration (denaturation) by the NPs, whereas the conformations of non-HB-GFs remain unaffected. We believe that this significant study will help structure-based design of therapeutic NPs to inhibit the functions of disease-causing proteins. From the Clinical Editor In this landmark paper by Arvizo and colleagues, the angiogenesis inhibitor effects of gold nanoparticles were investigated as the function of size and charge. This study will pave the way to the development of therapeutic NPs that inhibit the functions of pathogenic proteins.

Matrix Therapy with RGTA OTR4120 Improves Healing Time and Quality in Hairless Rats with Deep Second-Degree Burns

ReGeneraTing Agents (RGTAs) are biodegradable polymers engineered to mimic heparan-sulfate in the extracellular matrix of damaged tissue. RGTAs improve tissue healing in several animal models by stabilizing and protecting heparin-binding growth factors and matrix proteins. RGTA restores the normal matrix architecture and supports tissue regeneration. In this study, the authors evaluated the effects of RGTA on epidermal repair and dermal remodeling in a rat burn model. Deep second-degree burns were induced in 156 hairless rats, of which half (n = 78) received topical and intramuscular RGTA immediately after the burn followed by intramuscular RGTA weekly for 1 month. The controls (n = 78) received saline according to the same protocol. Rats were killed starting on each day of the first week and on days 14, 28, 60, 120, 240, and 365. The burns were evaluated by photography, histology, and immunohistochemistry. Coagulation necrosis involved the entire epidermis and superficial adnexa. Compared with the controls, speed of epidermal repair, as assessed between days 3 and 7 based on cell-layer number and anticytokeratin-14 staining, was faster in the RGTA group; and the zone of stasis, as assessed based on secondary vascular lesions in the dermis, was smaller. On day 7, reepithelialization was complete in both groups. On days 14 and 28, the remodeled dermal zone was smaller in the RGTA group. RGTA accelerated epidermal repair and protected the dermis from secondary effects of heat as quantified by zone-of-stasis size and extent of dermal remodeling.

Low-Molecular-Weight Heparin and Protamine-Based Polyelectrolyte Nano Complexes for Protein Delivery (A Review Articles)

We produced low-molecular-weight heparin/protamine micro (nano) particles (LMW-H/P MPs·NPs) as a carrier for heparin-binding growth factors (GFs), such as fibroblast growth factor (FGF)-2 and various GFs in platelet-rich plasma (PRP). A mixture of LMW-H (MW: approximately 5000 Da, 6.4 mg/ml) and protamine (MW: approximately 3000 Da, 10 mg/ml) at a ratio of 7:3 (vol:vol) yields a dispersion of micro (nano) particles (200 nm - 3 µm in diameter). The diluted LMW-H solution in saline (0.32 mg/ml) mixed with diluted protamine (0.5 mg/ml) at a ratio at 7:3 (vol:vol) resulted in soluble nanoparticles (approximately 100 nm in diameter). The generated NPs could be then stabilized by adding 2 mg/ml dextran (MW: 178-217 kDa) and remained soluble after lyophilization of dialyzed LMW-H /P NPs solution. The LMW-H/P MPs·NPs adsorb GFs, control their release, protect GFs and activate their biological activities. Furthermore, administration of GFs-containing F/P MPs·NPs exhibited significantly higher inductions of vascularization and fibrous tissue formation in vivo than GFs alone. LMW-H/P MPs·NPs can also efficiently bind to tissue culture plates and retain the binding of GFs. The LMW-H/P MPs·NP-coated matrix with various GFs or cytokines provided novel biomaterials that could control cellular activity such as proliferation and differentiation. Thus, LMW-H/P MPs·NPs are an excellent carrier for GFs and are a functional coating matrix for various kinds of cell cultures.

A [polycation:heparin] complex releases growth factors with enhanced bioactivity

Growth factors are potent molecules that regulate cell functions including survival, self renewal, differentiation and proliferation. High-efficacy delivery of growth factors will be a powerful tool for regenerative medicine. Decades of intense research have significantly advanced the field of controlled delivery. There is, however, still a great unmet need for new methods that can improve overall efficacy of growth factor delivery. Here, we report a new growth factor delivery vehicle formed by self assembly of heparin and a biocompatible polycation, poly(ethylene argininylaspartate diglyceride) (PEAD). Of the many heparin-binding growth factors, we chose FGF-2 and NGF to demonstrate the potential of the [PEAD:heparin] delivery vehicle. The delivery vehicle incorporates both growth factors with high efficiency, controls their release, maintains the bioactivity of FGF-2 and increases the bioactivity of NGF relative to bolus delivery. [PEAD:heparin] appears to be a promising delivery matrix for many heparin-binding growth factors and may lead to efficient growth factor delivery for a variety of diseases and disabilities.

A bioactive self-assembled membrane to promote angiogenesis

We report here on a bioactive hierarchically structured membrane formed by self-assembly. The membrane is formed with hyaluronic acid and peptide amphiphiles with binding affinity for heparin, and its hierarchical structure contains both an amorphous zone and a layer of fibrils oriented perpendicular to the membrane plane. The design of bioactivity is based on the potential ability to bind and slowly release heparin-binding growth factors. Human mesenchymal stem cells (hMSCs) seeded on these membranes attached and remained viable. Basic fibroblast growth factor (FGF2) and vascular endothelial growth factor (VEGF) were incorporated within the membrane structure prior to self-assembly and released into media over a prolonged period of time (14 days). Using the chicken chorioallantoic membrane (CAM) assay, we also found that these membranes induced a significant and rapid enhancement of angiogenesis relative to controls.

Soluble perlecan domain i enhances vascular endothelial growth factor-165 activity and receptor phosphorylation in human bone marrow endothelial cells

BackgroundImmobilized recombinant perlecan domain I (PlnDI) binds and modulates the activity of heparin-binding growth factors, in vitro. However, activities for PlnDI, in solution, have not been reported. In this study, we assessed the ability of soluble forms to modulate vascular endothelial growth factor-165 (VEGF165) enhanced capillary tube-like formation, and VEGF receptor-2 phosphorylation of human bone marrow endothelial cells, in vitro.ResultsIn solution, PlnDI binds VEGF165 in a heparan sulfate and pH dependent manner. Capillary tube-like formation is enhanced by exogenous PlnDI; however, PlnDI/VEGF165 mixtures combine to enhance formation beyond that stimulated by either PlnDI or VEGF165 alone. PlnDI also stimulates VEGF receptor-2 phosphorylation, and mixtures of PlnDI/VEGF165 reduce the time required for peak VEGF receptor-2 phosphorylation (Tyr-951), and increase Akt phosphorylation. PlnDI binds both immobilized neuropilin-1 and VEGF receptor-2, but has a greater affinity for neuropilin-1. PlnDI binding to neuropilin-1, but not to VEGF receptor-2 is dependent upon the heparan sulfate chains adorning PlnDI. Interestingly, the presence of VEGF165 but not VEGF121 significantly enhances PlnDI binding to Neuropilin-1 and VEGF receptor-2.ConclusionsOur observations suggest soluble forms of PlnDI are biologically active. Moreover, PlnDI heparan sulfate chains alone or together with VEGF165 can enhance VEGFR-2 signaling and angiogenic events, in vitro. We propose PlnDI liberated during basement membrane or extracellular matrix turnover may have similar activities, in vivo.