A Wnt-mediated phenotype switch along the epithelial-mesenchymal axis defines resistance and invasion downstream of ionising radiation in oral squamous cell carcinoma.

BACKGROUND: Dynamic transitions of tumour cells along the epithelial-mesenchymal axis are important in tumorigenesis, metastasis and therapy resistance. METHODS: In this study, we have used cell lines, 3D spheroids and tumour samples in a variety of cell biological and transcriptome analyses to highlight the cellular and molecular dynamics of OSCC response to ionising radiation. RESULTS: Our study demonstrates a prominent hybrid epithelial-mesenchymal state in oral squamous cell carcinoma cells and tumour samples. We have further identified a key role for levels of E-cadherin in stratifying the hybrid cells to compartments with varying levels of radiation response and radiation-induced epithelial-mesenchymal transition. The response to radiation further entailed the generation of a new cell population with low expression levels of E-cadherin, and positive for Vimentin (ECADLow/Neg-VIMPos), a phenotypic signature that showed an enhanced capacity for radiation resistance and invasion. At the molecular level, transcriptome analysis of spheroids in response to radiation showed an initial burst of misregulation within the first 30 min that further declined, although still highlighting key alterations in gene signatures. Among others, pathway analysis showed an over-representation for the Wnt signalling pathway that was further confirmed to be functionally involved in the generation of ECADLow/Neg-VIMPos population, acting upstream of radiation resistance and tumour cell invasion. CONCLUSION: This study highlights the functional significance and complexity of tumour cell remodelling in response to ionising radiation with links to resistance and invasive capacity. An area of less focus in conventional radiotherapy, with the potential to improve treatment outcomes and relapse-free survival.

Uterine cells are recruited to the infarcted heart and improve cardiac outcomes in female rats.

We evaluated the hypothesis that uterine cells home to the heart after injury and improve cardiac outcomes. Premenopausal women have fewer cardiovascular complications than age-matched men, but the mechanisms responsible for this protection have not been conclusively identified. Hysterectomy was performed in young female rats (leaving the ovaries intact), and 7 days later the left coronary artery was ligated to produce a myocardial infarction (MI). Cardiac function at 28 days post-MI was measured using echocardiography. Fractional shortening was best in non-hysterectomized (non-Hx) females and lower in both Hx females and males. Uteri were then removed from GFP rats and heterotopically transplanted into non-GFP recipients to investigate homing of uterine cells to the infarcted myocardium. Seven days later, the uterine transplant recipients underwent coronary ligation. GFP(+) cells were found in the recipient hearts 7 days after MI and persisted for 6 months. Confocal analysis showed that homed uterine cells were located around blood vessels, suggesting their involvement in neovascularization. We then evaluated uterine cell transplantation by intravenously injecting GFP(+) uterine cells into Hx females immediately after MI. These GFP(+) cells were found to home to the injured myocardium, stimulate angiogenesis, improve cardiac function, and increase survival. This study demonstrates that uterine cells can home to the injured myocardium, enhance tissue repair, and prevent cardiac dysfunction. Uterine cells may play a role in the prevention of cardiovascular complications in females.

Role of PDGF-A/B Ligands in Cardiac Repair After Myocardial Infarction.

Platelet-derived growth factors (PDGFs) are powerful inducers of cellular mitosis, migration, angiogenesis, and matrix modulation that play pivotal roles in the development, homeostasis, and healing of cardiac tissues. PDGFs are key signaling molecules and important drug targets in the treatment of cardiovascular disease as multiple researchers have shown that delivery of recombinant PDGF ligands during or after myocardial infarction can reduce mortality and improve cardiac function in both rodents and porcine models. The mechanism involved cannot be easily elucidated due to the complexity of PDGF regulatory activities, crosstalk with other protein tyrosine kinase activators, and diversity of the pathological milieu. This review outlines the possible roles of PDGF ligands A and B in the healing of cardiac tissues including reduced cell death, improved vascularization, and improved extracellular matrix remodeling to improve cardiac architecture and function after acute myocardial injury. This review may highlight the use of recombinant PDGF-A and PDGF-B as a potential therapeutic modality in the treatment of cardiac injury.

Senescent impairment in synergistic cytokine pathways that provide rapid cardioprotection in the rat heart.

Pretreatment of rodent hearts with platelet-derived growth factor (PDGF)-AB decreases myocardial injury after coronary occlusion. However, PDGF-AB cardioprotection is diminished in older animals, suggesting that downstream elements mediating and/or synergizing the actions of PDGF-AB may be limited in aging cardiac vasculature. In vitro PDGF-AB induced vascular endothelial growth factor (VEGF) and angiopoietin (Ang)-2 expression in 4-mo-old rat cardiac endothelial cells, but not in 24-mo-old heart cells. In vivo injection of young hearts with PDGF-AB increased densities of microvessels staining for VEGF and its receptor, Flk-1, and Ang-2 and its receptor, Tie-2, as well as PDGF receptor (PDGFR)-alpha. In older hearts, PDGF-AB-mediated induction was primarily limited to PDGFR-alpha. Studies in a murine cardiac transplantation model demonstrated that synergist interactions of PDGF-AB plus VEGF plus Ang-2 (PVA) provided an immediate restoration of senescent cardiac vascular function. Moreover, PVA injection in young rat hearts, but not PDGF-AB alone or other cytokine combinations, at the time of coronary occlusion suppressed acute myocardial cell death by >50%. However, PVA also reduced the extent of myocardial infarction with an age-associated cardioprotective benefit (4-mo-old with 45% reduction vs. 24-mo-old with 24%; P < 0.05). These studies showed that synergistic cytokine pathways augmenting the actions of PDGF-AB are limited in older hearts, suggesting that strategies based on these interactions may provide age-dependent clinical cardiovascular benefit.

c-Kit function is necessary for in vitro myogenic differentiation of bone marrow hematopoietic cells.

In recent years, the differentiation of bone marrow cells (BMCs) into myocytes has been extensively investigated, but the findings remain inconclusive. The purpose of this study was to determine the conditions necessary to induce myogenic differentiation in short-term cultures of adult BMCs, and to identify the BMC subpopulation responsible for this phenomenon. We report that high-density cultures of murine hematopoietic BMCs gave rise to spontaneous beating cell clusters in the presence of vascular endothelial and fibroblast growth factors. These clusters originated from c-kit(pos) cells. The formation of the clusters could be completely blocked by adding a c-kit/tyrosine kinase inhibitor, Gleevec (imatinib mesylate; Novartis International, Basel, Switzerland, http://www.novartis.com), to the culture. Cluster formation was also blunted in BMCs from c-kit-deficient (Kit(W)/Kit(W-v)) mice. Clustered cells expressed cardiomyocyte-specific transcription factor genes Gata-4 and Nkx2.5, sarcomeric proteins beta-MHC and MLC-2v, and ANF and connexin-43. Immunostaining revealed alpha-sarcomeric actinin expression in more than 90% of clustered cells. Under electron microscopy, the clustered cells exhibited a sarcomeric myofiber arrangement and z-bands. This study defines the microenvironment required to achieve a reproducible in vitro model of beating, myogenic cell clusters. This model could be used to examine the mechanisms responsible for the postnatal myogenic differentiation of BMCs. Our results identify c-kit(pos) bone marrow hematopoietic cells as the source of the myogenic clusters.

Vascularity during wound maturation correlates with fragmentation of serum albumin but not ceruloplasmin, transferrin, or haptoglobin.

Reduced vascularity during wound maturation is mediated by endothelial apoptosis. Albumin has an anti-apoptotic activity for endothelium, which increases up to 100-fold on albumin fragmentation (AF). We now report that levels of AF correlate with changing vascularity during wound maturation. Both scarring and adipogenic wound-healing models were established in mice. Western blots of granulation tissue revealed AF concurrent with periods of high vascularity as determined by thin-section microscopy, with reduced AF on wound maturation (p<0.02). In profiling AF, the levels of 27.5 and 39 kDa fragments were reduced on maturation of both scarring and adipogenic wounds (p<0.005), as were the levels of an additional 17.5 kDa fragment prominent only in adipogenic wounds (p<0.001). A 49 kDa albumin fragment was found to be reduced during maturation of scarring (p<0.001) but not adipogenic wounds. For comparison, we probed for transferrin, ceruloplasmin, and haptoglobin fragmentation on the basis that like albumin, these are considered acute-phase transport proteins. Minimal fragmentation of transferrin and ceruloplasmin was seen, along with partial dissociation of haptoglobin subunits, but these did not correlate with AF or vascularity. Our findings are consistent with a role for AF in regulating granulation tissue vascularity during healing.

Platelet-derived growth factor-AB improves scar mechanics and vascularity after myocardial infarction.

Therapies that target scar formation after myocardial infarction (MI) could prevent ensuing heart failure or death from ventricular arrhythmias. We have previously shown that recombinant human platelet-derived growth factor-AB (rhPDGF-AB) improves cardiac function in a rodent model of MI. To progress clinical translation, we evaluated rhPDGF-AB treatment in a clinically relevant porcine model of myocardial ischemia-reperfusion. Thirty-six pigs were randomized to sham procedure or balloon occlusion of the proximal left anterior descending coronary artery with 7-day intravenous infusion of rhPDGF-AB or vehicle. One month after MI, rhPDGF-AB improved survival by 40% compared with vehicle, and cardiac magnetic resonance imaging showed left ventricular (LV) ejection fraction improved by 11.5%, driven by reduced LV end-systolic volumes. Pressure volume loop analyses revealed improved myocardial contractility and energetics after rhPDGF-AB treatment with minimal effect on ventricular compliance. rhPDGF-AB enhanced angiogenesis and increased scar anisotropy (high fiber alignment) without affecting overall scar size or stiffness. rhPDGF-AB reduced inducible ventricular tachycardia by decreasing heterogeneity of the ventricular scar that provides a substrate for reentrant circuits. In summary, we demonstrated that rhPDGF-AB promotes post-MI cardiac wound repair by altering the mechanics of the infarct scar, resulting in robust cardiac functional improvement, decreased ventricular arrhythmias, and improved survival. Our findings suggest a strong translational potential for rhPDGF-AB as an adjunct to current MI treatment and possibly to modulate scar in other organs.

Neural microvascular pericytes contribute to human adult neurogenesis.

Consistent adult neurogenic activity in humans is observed in specific niches within the central nervous system. However, the notion of an adult neurogenic niche is challenged by accumulating evidence for ectopic neurogenic activity in other cerebral locations. Herein we interface precision of ultrastructural resolution and anatomical simplicity of accessible human dental pulp neurogenic zone to address this conflict. We disclose a basal level of adult neurogenic activity characterized by glial invasion of terminal microvasculature followed by release of individual platelet-derived growth factor receptor-ß mural pericytes and subsequent reprogramming into NeuN+ local interneurons. Concomitant angiogenesis, a signature of adult neurogenic niches, accelerates the rate of neurogenesis by amplifying release and proliferation of the mural pericyte population by ≈10-fold. Subsequent in vitro and in vivo experiments confirmed gliogenic and neurogenic capacities of human neural pericytes. Findings foreshadow the bimodal nature of the glio-vascular assembly where pericytes, under instruction from glial cells, can stabilize the quiescent microvasculature or enrich local neuronal microcircuits upon differentiation.

Single-cell expression profiling reveals dynamic flux of cardiac stromal, vascular and immune cells in health and injury.

Besides cardiomyocytes (CM), the heart contains numerous interstitial cell types which play key roles in heart repair, regeneration and disease, including fibroblast, vascular and immune cells. However, a comprehensive understanding of this interactive cell community is lacking. We performed single-cell RNA-sequencing of the total non-CM fraction and enriched (Pdgfra-GFP+) fibroblast lineage cells from murine hearts at days 3 and 7 post-sham or myocardial infarction (MI) surgery. Clustering of >30,000 single cells identified >30 populations representing nine cell lineages, including a previously undescribed fibroblast lineage trajectory present in both sham and MI hearts leading to a uniquely activated cell state defined in part by a strong anti-WNT transcriptome signature. We also uncovered novel myofibroblast subtypes expressing either pro-fibrotic or anti-fibrotic signatures. Our data highlight non-linear dynamics in myeloid and fibroblast lineages after cardiac injury, and provide an entry point for deeper analysis of cardiac homeostasis, inflammation, fibrosis, repair and regeneration.

c-kit dysfunction impairs myocardial healing after infarction.

BACKGROUND: We hypothesized that c-kit receptor function in the bone marrow is important for facilitating healing, leading to efficient cardiac repair after myocardial infarction (MI). METHODS AND RESULTS: We used Kit(W)/Kit(W-v) c-kit mutant mice and their wild-type littermates to assess the importance of c-kit function in cardiac remodeling after coronary ligation. We found that mutant mice developed 1.6-fold greater ventricular dilation (P=0.008) attributable to a 1.3-fold greater infarct expansion by day 14 after MI (P=0.01). The number of proliferating smooth muscle alpha-actin expressing cells was 1.8-fold lower in mutant mice at day 3 (P<0.01), resulting in a 1.6 to 1.8-fold reduction in total regional nonvascular smooth muscle alpha-actin expressing cells by both microscopy and flow cytometry (P<0.001 for both). This decrease was accompanied by a 1.4-fold reduction in the number of CD31 expressing blood vessels (P<0.05). Prior transplantation of wild-type bone marrow cells into mutant mice rescued the efficient establishment of vessel-rich repair tissue by inducing a 1.5-fold increase in nonvascular smooth muscle alpha-actin expressing cells and CD31 expressing blood vessels (P<0.05 for both). The increased recruitment of cells into the infarct region in the chimeric mice was associated with reduced infarct expansion (P<0.03) compared to wild-type levels. CONCLUSIONS: Bone marrow c-kit function critically impacts the myofibroblast repair response in infarcted hearts. Interventions that increase the infiltration of c-kit+ cells to the infarcted heart may potentiate this endogenous repair response, prevent infarct expansion, and improve the recovery of cardiac function after MI.

BDNF-mediated enhancement of inflammation and injury in the aging heart.

Aging is associated with shifts in autocrine and paracrine pathways in the cardiac vasculature that may contribute to the risk of cardiovascular disease in older persons. To elucidate the molecular basis of these changes in vivo, phage-display biopanning of 3- and 18-mo-old mouse hearts was performed that identified peptide epitopes with homology to brain-derived neurotrophic factor (BDNF) in old but not young phage pools. Quantification of cardiac phage binding by titration and immunostaining after injection with BDNF-like phage identified a twofold increased density of the BDNF receptor, truncated Trk B, in the aging hearts. Studies focused on the receptor ligand using a rat model of transient myocardial ischemia revealed increases in cardiac BDNF associated with local mononuclear infiltrates in 24- but not 4-mo-old rats. To investigate these changes, both 4- and 24-mo-old rat hearts were treated with intramyocardial injections of BDNF (or PBS control), demonstrating significant inflammatory increases with activated macrophage (ED1+) in BDNF-treated aging hearts compared with aging controls and similarly treated young hearts. Additional studies with permanent coronary occlusion following intramyocardial growth factor pretreatment revealed that BDNF significantly increased the extent of myocardial injury in older rat hearts (BDNF 35 +/- 10% vs. PBS 16.2 +/- 7.9% left ventricular injury; P < 0.05) without affecting younger hearts (BDNF 15 +/- 5.1% vs. PBS 14.5 +/- 6.0% left ventricular injury). Overall, these studies suggest that age-associated changes in BDNF-Trk B pathways may predispose the aging heart to increased injury after acute myocardial infarction and potentially contribute to the enhanced severity of cardiovascular disease in older individuals.

Platelet-derived growth factor-AB promotes the generation of adult bone marrow-derived cardiac myocytes.

The directed generation of cardiac myocytes from endogenous stem cells offers the potential for novel therapies for cardiovascular disease. To facilitate the development of such approaches, we sought to identify and exploit the pathways directing the generation of cardiac myocytes from adult rodent bone marrow cells (BMCs). In vitro cultures supporting the spontaneous generation of functional cardiac myocytes from murine BMCs demonstrated induced expression of platelet-derived growth factor (PDGF)-A and -B isoforms with alpha- and beta-myosin heavy chains as well as connexin43. Supplementation of PDGF-AB speeded the kinetics of myocyte development in culture by 2-fold. In a rat heart, myocardial infarction pretreatment model PDGF-AB also promoted the derivation of cardiac myocytes from BMCs, resulting in a significantly greater number of islands of cardiac myocyte bundles within the myocardial infarction scar compared with other treatment groups. However, gap junctions were detected only between the cardiac myocytes receiving BMCs alone, but not BMCs injected with PDGF-AB. Echocardiography and exercise testing revealed that the functional improvement of hearts treated with the combination of BMCs and PDGF-AB was no greater than with injections of BMCs or PDGF-AB alone. These studies demonstrated that PDGF-AB enhances the generation of BMC-derived cardiac myocytes in rodent hearts, but suggest that alterations in cellular patterning may limit the functional benefit from the combined injection of PDGF-AB and BMCs. Strategies based on the synergistic interactions of PDGF-AB and endogenous stem cells will need to maintain cellular patterning in order to promote the restoration of cardiac function after acute coronary occlusion.

Hox-Mediated Spatial and Temporal Coding of Stem Cells in Homeostasis and Neoplasia.

Hox genes are fundamental components of embryonic patterning and morphogenesis with expression persisting into adulthood. They are also implicated in the development of diseases, particularly neoplastic transformations. The tight spatio-temporal regulation of Hox genes in concordance with embryonic patterning is an outstanding feature of these genes. In this review we have systematically analyzed Hox functions within the stem/progenitor cell compartments and asked whether their temporo-spatial topography is retained within the stem cell domain throughout development and adulthood. In brief, evidence support involvement of Hox genes at several levels along the stem cell hierarchy, including positional identity, stem cell self-renewal, and differentiation. There is also strong evidence to suggest a role for Hox genes during neoplasia. Although fundamental questions are yet to be addressed through more targeted and high- throughput approaches, existing evidence suggests a central role for Hox genes within a continuum along the developmental axes persisting into adult homeostasis and disease.

Platelet-Derived Growth Factor Receptor Alpha as a Marker of Mesenchymal Stem Cells in Development and Stem Cell Biology.

Three decades on, the mesenchymal stem cells (MSCs) have been intensively researched on the bench top and used clinically. However, ambiguity still exists in regard to their anatomical locations, identities, functions, and extent of their differentiative abilities. One of the major impediments in the quest of the MSC research has been lack of appropriate in vivo markers. In recent years, this obstacle has been resolved to some degree as PDGFRα emerges as an important mesenchymal stem cell marker. Accumulating lines of evidence are showing that the PDGFRα (+) cells reside in the perivascular locations of many adult interstitium and fulfil the classic concepts of MSCs in vitro and in vivo. PDGFRα has long been recognised for its roles in the mesoderm formation and connective tissue development during the embryogenesis. Current review describes the lines of evidence regarding the role of PDGFRα in morphogenesis and differentiation and its implications for MSC biology.

Uterine-derived stem cells reconstitute the bone marrow of irradiated mice.

Hematopoietic stem cells (HSCs) can be found in several tissues of mesodermal origin. Uterine tissue contains stem cells and can regenerate during each menstrual cycle with robust new tissue formation. Stem cells may play a role in this regenerative potential. Here, we report that transplantation of cells isolated from murine uterine tissue can rescue lethally irradiated mice and reconstitute the major hematopoietic lineages. Donor cells can be detected in the blood and hematopoietic tissues such as spleen and bone marrow (BM) of recipient mice. Uterine tissue contains a significant percentage of cells that are Sca-1(+), Thy 1.2(+), or CD45(+) cells, and uterine cells (UCs) were able to give rise to hematopoietic colonies in methylcellulose. Using secondary reconstitution, a key test for hematopoietic potential, we found that the UCs exhibited HSC-like reconstitution of BM and formation of splenic nodules. In a sensitive assay for cell fusion, we used a mixture of cells from Cre and loxP mice for reconstitution and demonstrated that hematopoietic reconstitution by UCs is not a function of fusion with donor BM cells. We also showed that the hematopoietic potential of the uterine tissue was not a result of BM stem cells residing in the uterine tissue. In conclusion, our data provide novel evidence that cells isolated from mesodermal tissues such as the uterus can engraft into the hematopoietic system of irradiated recipients and give rise to multiple hematopoietic lineages. Thus, uterine tissue could be considered an important source of stem cells able to support hematopoiesis.

Phage display identification of age-associated TNFalpha-mediated cardiac oxidative induction.

Age-associated alterations in the actions of tumor necrosis factor-alpha (TNFalpha) in the heart with impaired cardioprotective pathways and enhanced apoptotic induction may contribute to the increased severity of cardiovascular pathology in older persons. To identify the molecular events mediating these changes in the microvasculature of the aging rodent heart, the biochemical properties of in vivo phage-display cyclic peptide cardiac biopanning were studied. Analysis of individual amino acid positions revealed that the center of the peptide motif (amino acid position 4) had a significantly higher frequency of aromatic amino acid side chains in phage homing to the old hearts compared with young controls (18 mo old, 11% vs. 3 mo old, 3%, P < 0.05). This subset of phage motifs revealed an age-associated homology with oxidoreductase enzymes (homology: 18 mo, 7/7; 3 mo, 0/2), suggesting the substrates and/or binding sites of these enzymes are increased in the aging hearts. Immunostaining for the oxidoreductase substrate 4-hydroxy-2-nonenal (HNE), a cardiotoxic lipid peroxidation product, demonstrated a twofold higher density of HNE(+) cells in PBS-treated hearts of old mice (18 mo) compared with young controls (3 mo) (18 mo, 3.2 +/- 2.8 vs. 3 mo, 1.0 +/- 0.9 cells/HPF, P < 0.05). Moreover, intracardiac injection of TNFalpha resulted in a significantly greater increase in HNE staining in the old hearts (18 mo, 16.9 +/- 13.8 vs. 3 mo, 9.1 +/- 6.0 cells/HPF, P < 0.05). Overall, these studies demonstrate that aging-associated alterations in TNFalpha-mediated pathways with induction of reactive oxidative species and changes in vascular surface binding sites may contribute mechanistically to the increased cardiovascular pathology of the aging heart.

Translation of PDGF cardioprotective pathways.

Vascular function in the aging heart is impaired and may underlie the increased morbidity and mortality associated with ischemic heart disease in older individuals. This vascular dysfunction is due, in part, to impairment of platelet derived growth factor (PDGF)-mediated pathways in senescent cardiac microvascular endothelial cells. Restoration of these pathways by intramyocardial injection of growth factor transiently rescues senescent cardiac angiogenesis. Longer-term reconstitution can be achieved experimentally by transplantation of young bone marrow-derived stem cells to promote senescent cardiac angiogenic function in the murine host. Moreover, enhancement of PDGF pathways is cardioprotective, markedly reducing the extent of myocardial injury following coronary occlusion. The clinical translation of these findings for treatment of ischemic heart diseases must overcome the limitation of the proatherosclerotic actions of PDGF, as well as the generation of autologous stem/precursor cell approaches, for the aging cardiovascular system. Strategies targeting growth factor and/or stem-cell homing to gene products downstream of PDGF in the cardiac microvasculature may provide positive feedback loops to enhance cardiac angiogenesis and protection from myocardial infarction and may offer a foundation for developing novel therapies for the prevention and treatment of cardiovascular disease associated with aging.

Platelet-derived growth factor improves cardiac function in a rodent myocardial infarction model.

OBJECTIVES: The translation of cardioprotective therapies for myocardial infarction requires a preclinical demonstration of improved cardiovascular function following acute coronary occlusion. We previously showed that pretreatment of rodent hearts with platelet-derived growth factor (PDGF) promotes angiogenesis and decreases the extent of myocardial injury measured by histology. The present study aimed to determine the correlation of these histological findings with noninvasive measures of improvement in cardiac function. METHODS: Rats were treated with intramyocardial injections of PDGF (100 ng) or phosphate buffer solution (PBS) (n = 6 per group) 24 h prior to acute, permanent ligation of the left anterior descending artery and the extent of myocardial injury was assessed by Masson's trichrome staining 14 days later. To assess the physiological effects of PDGF pretreatment after coronary occlusion, cardiac function was assessed noninvasively by electrocardiography, exercise testing and echocardiography and correlated with direct histological measures. RESULTS: Physiological studies demonstrated that PDGF resulted in lower ST-segment elevation at the time of coronary occlusion (0.12 +/- 0.02 mV above baseline) than in PBS control rats (0.35 +/- 0.05 mV; P < 0.05). Exercise testing 14 days after coronary occlusion revealed that PDGF pretreatment resulted in faster maximal exercise speeds (28.54 +/- 3.98 m/min) than in control rats (24.98 +/- 3.13 m/min; P < 0.05). Echocardiography also revealed that the left ventricular factional shortening in the PDGF-pretreated rats was significantly greater (18.47 +/- 12.21%) than in control animals (4.91 +/- 7.21%; P<0.05). CONCLUSIONS: These studies demonstrate that PDGF pretreatment improves cardiac function following acute coronary occlusion. Strategies based on the cardioprotective actions of PDGF may provide a significant advance in the treatment of myocardial infarction.

Adult cardiac stem cells--where do we go from here?

A potential treatment for cardiovascular disease involves the transplantation of a patient's bone marrow stem cells into the heart of that same patient. In order to maximize the potential benefits to select patient populations, the continued clinical development of this technology will require a comprehensive understanding of the role(s) of the transplanted cells in the repair of damaged heart tissue as well as an understanding of which types of cardiac injury can be repaired by this approach. The widespread application of cardiovascular stem cell therapies, however, will likely be based on pharmacological approaches to enhance the capacity of endogenous bone marrow stem cells to provide for the replacement of cardiac muscle and vascular cells after myocardial injury.

SAOS-2 osteosarcoma cells bind fibroblasts via ICAM-1 and this is increased by tumour necrosis factor-α.

We recently reported exchange of membrane and cytoplasmic markers between SAOS-2 osteosarcoma cells and human gingival fibroblasts (h-GF) without comparable exchange of nuclear markers, while similar h-GF exchange was seen for melanoma and ovarian carcinoma cells. This process of "cellular sipping" changes phenotype such that cells sharing markers of both SAOS-2 and h-GF have morphology intermediate to that of either cell population cultured alone, evidencing increased tumour cell diversity without genetic change. TNF-α increases cellular sipping between h-GF and SAOS-2, and we here study binding of SAOS-2 to TNF-α treated h-GF to determine if increased cellular sipping can be accounted for by cytokine stimulated SAOS-2 binding. More SAOS-2 bound h-GF pe-seeded wells than culture plastic alone (p<0.001), and this was increased by h-GF pre-treatment with TNF-α (p<0.001). TNF-α stimulated binding was dose dependent and maximal at 1.16 nM (p<0.05) with no activity below 0.006 nM. SAOS-2 binding to h-GF was independent of serum, while the lipopolysaccharide antagonist Polymyxin B did not affect results, and TNF-α activity was lost on boiling. h-GF binding of SAOS-2 started to increase after 30min TNF-α stimulation and was maximal by 1.5 hr pre-treatment (p<0.001). h-GF retained maximal binding up to 6 hrs after TNF-α stimulation, but this was lost by 18 hrs (p<0.001). FACS analysis demonstrated increased ICAM-1 consistent with the time course of SAOS-2 binding, while antibody against ICAM-1 inhibited SAOS-2 adhesion (p<0.04). Pre-treating SAOS-2 with TNF-α reduced h-GF binding to background levels (p<0.003), and this opposite effect to h-GF cytokine stimulation suggests that the history of cytokine exposure of malignant cells migrating across different microenvironments can influence subsequent interactions with fibroblasts. Since cytokine stimulated binding was comparable in magnitude to earlier reported TNF-α stimulated cellular sipping, we conclude that TNF-α stimulated cellular sipping likely reflects increased SAOS-2 binding as opposed to enhanced exchange mechanisms.