Keiko Funa

Transplantation of Human Embryonic Stem Cell‐Derived Cells to a Rat Model of Parkinson's Disease: Effect of In Vitro Differentiation on Graft Survival and Teratoma Formation

Human embryonic stem cells (hESCs) have been proposed as a source of dopamine (DA) neurons for transplantation in Parkinsons disease (PD). We have investigated the effect of in vitro predifferentiation on in vivo survival and differentiation of hESCs implanted into the 6‐OHDA (6‐hydroxydopamine)‐lesion rat model of PD. The hESCs were cocultured with PA6 cells for 16, 20, or 23 days, leading to the in vitro differentiation into DA neurons. Grafted hESC‐derived cells survived well and expressed neuronal markers. However, very few exhibited a DA neuron phenotype. Reversal of lesion‐induced motor deficits was not observed. Rats grafted with hESCs predifferentiated in vitro for 16 days developed severe teratomas, whereas most rats grafted with hESCs predifferentiated for 20 and 23 days remained healthy until the end of the experiment. This indicates that prolonged in vitro differentiation of hESCs is essential for preventing formation of teratomas.

The L45 loop in type I receptors for TGF-β family members is a critical determinant in specifying Smad isoform activation

Transforming growth factor‐β (TGF‐β) and bone morphogenetic proteins (BMPs) signal via distinct type I and type II receptors and Smad proteins. A nine amino acid sequence between kinase subdomains IV and V in type I receptors, termed the L45 loop, has been shown to be important in conferring signalling specificity. We examined the responses of a mutant TGF‐β type I receptor (TβR‐I) and a mutant BMPR‐IB, in which the L45 regions of these two receptors were exchanged. Swapping the four amino acid residues that are different in BMPR‐IB for those in TβR‐I, and vice versa, switched their type I receptor‐restricted Smad activation and specificity in transcriptional responses. These studies identify the L45 loop regions in type I receptors as critical determinants in specifying Smad isoform activation.

Platelet-Derived Growth Factor is Angiogenic In Vivo

PDGF receptors have recently been found to be expressed in microvascular endothelium in vivo under circumstances of endothelial cell activation and angiogenesis suggesting that PDGF may have a direct effect on endothelial cells. We have tested the angiogenic activity of PDGF-AA and -BB homodimers in the chick chorioallantoic membrane in vivo. PDGF-BB was found to consistently induce an angiogenic response whereas PDGF-AA was less active. Morphological analyses revealed that there was little inflammation associated with this response but an increase in vessel density suggested a direct effect of PDGF on embryonic chorioallantoic endothelial cells. In vitro, PDGF-BB was found to be more potent than PDGF-AA in stimulating the chemotaxis of rat brain capillary endothelial cells. This is consistent with a direct effect of PDGF on endothelial cells. Thus, this novel angiogenic activity of PDGF has implications for several developmental and pathological events in which PDGF, particularly the B-chain, is expressed.

Apoptosis and expression of Bcl-2 after compression trauma to rat spinal cord.

We have evaluated by in situ nick-end labeling the presence of apoptotic cells in the spinal cord of rats with compression injury at the level of Th8–9, of mild, moderate, and severe degrees resulting in no neurologic deficit, reversible paraparesis, and paraplegia, respectively. Rats with compression injury surviving 4 or 9 days showed apoptotic glial cells in the longitudinal tracts of the Th8–9, the cranial Th7, and the caudal Th10 segments. The apoptotic cells were most frequently observed in Th7. They did not express glial fibrillar acidic protein (GFAP) and their morphology was compatible with that of oligodendrocytes. Neurons of the gray matter did not present signs of apoptosis. In addition, we studied the immunohisto-chemical expression of Bcl-2, an endogenous inhibitor of apoptosis. Compression induced Bcl-2 immunoreactivity in axons of the long tracts, particularly after moderate and severe compression and 1-day survival. Neurons of dorsal root ganglia were immunoreactive but the neurons of the spinal cord were unstained. The accumulation, presumably caused by arrested axonal transport in sensory pathways, was absent in rats surviving 9 days. In conclusion, compression trauma to rat spinal cord induces signs of apoptosis in glial cells, presumably oligodendrocytes of the long tracts. This may induce delayed myelin degeneration after trauma to the spinal cord. Bcl-2 does not seem to be upregulated in oligodendrocytes

Angiogenic factors FGF2 and PDGF-BB synergistically promote murine tumor neovascularization and metastasis

Tumors produce multiple growth factors, but little is known about the interplay between various angiogenic factors in promoting tumor angiogenesis, growth, and metastasis. Here we show that 2 angiogenic factors frequently upregulated in tumors, PDGF-BB and FGF2, synergistically promote tumor angiogenesis and pulmonary metastasis. Simultaneous overexpression of PDGF-BB and FGF2 in murine fibrosarcomas led to the formation of high-density primitive vascular plexuses, which were poorly coated with pericytes and VSMCs. Surprisingly, overexpression of PDGF-BB alone in tumor cells resulted in dissociation of VSMCs from tumor vessels and decreased recruitment of pericytes. In the absence of FGF2, capillary ECs lacked response to PDGF-BB. However, FGF2 triggers PDGFR-alpha and -beta expression at the transcriptional level in ECs, which acquire hyperresponsiveness to PDGF-BB. Similarly, PDGF-BB-treated VSMCs become responsive to FGF2 stimulation via upregulation of FGF receptor 1 (FGFR1) promoter activity. These findings demonstrate that PDGF-BB and FGF2 reciprocally increase their EC and mural cell responses, leading to disorganized neovascularization and metastasis. Our data suggest that intervention of this non-VEGF reciprocal interaction loop for the tumor vasculature could be an important therapeutic target for the treatment of cancer and metastasis.

Expression of Vascular Endothelial Growth Factor (VEGF) and its Receptors (Flt-1 and Flk-1) following Permanent and Transient Occlusion of the Middle Cerebral Artery in the Rat

Vascular endothelial growth factor (VEGH) is a known endothelial mitogen and a potent enhancer of vascular permeability although its role in focal cerebral ischemia is still not completely understood. The present report describes the immunohistochemical distribution of VEGF and its 2 receptors, Flt-1 and Flk-1 at day 1 and 3 following permanent and transient middle cerebral artery occlusion (MCAO) in the rat. A bilateral increase in VEGF immunoreactivity, Particularly in neurons and blood vessels, was seen in both the experimental designs by day 1. By day 3, the immunoreactivity was restricted chiefly to the lesion side, where reaction was most prominent in the border zones of the infarcts. Immunoreaction to VEGF was more pronounced in cases of permanent MCAO than in transient MCAO.Flt-1 reaction was increased in neurons, gilal and endothelial cells after both transient and permanent MCAO. Immunoreactivity to Flk-1 was prominent in glial cells and was present to some extent in endothelial cells. These findings indicate an early upregulation of VEGF and its receptors after permanent as well as transient focal cerebral ischemia in the rat.

Noggin is required for induction of the hair follicle growth phase in postnatal skin

During postnatal development, the hair follicle (HF) shows cyclic activity with periods of relative resting, active growth (anagen), and regression. We demonstrate that similar to the HF induction in embryonic skin, initiation of a new hair growth phase in postnatal skin requires neutralization of the inhibitory activity of bone morphogenetic protein 4 (BMP4) by the BMP antagonist noggin. In the resting HF, BMP4 mRNA predominates over noggin in the epithelium and mesenchyme, and the BMP receptor IA is prominently expressed in the follicular germ. Anagen development is accompanied by down‐regulation of the BMP4 and increased noggin mRNA in the HF. Furthermore, administration of noggin protein induces new hair growth phase in postnatal telogen skin in vivo. In contrast, BMP4 induces selective arrest of anagen development in the non‐tylotrich (secondary) HF. As a hair growth inducer, noggin increases Shh mRNA in the HF whereas BMP4 down‐regulates Shh. This suggests that modulation of BMP4 signaling by noggin is essential for hair growth phase induction in postnatal skin and that the hair growth‐inducing effect of noggin is mediated, at least in part, by Shh.—Botchkarev, V. A., Botchkareva, N. V., Nakamura, M., Huber, O., Funa, K., Lauster, R., Paus, R., Gilchrest, B. A. Noggin is required for induction of the hair follicle growth phase in postnatal skin. FASEB J. 15, 2205–2214 (2001)

Platelet-derived growth factor promotes survival of rat and human mesencephalic dopaminergic neurons in culture

SummaryThe effect of two isoforms of platelet-derived growth factor (PDGF), PDGF-AA and PDGF-BB, was tested on dissociated cell cultures of ventral mesencephalon from rat and human embryos. PDGF-BB but not PDGF-AA reduced the progressive loss of tyrosine hydroxylase- (TH)-positive neurons in rat and human cell cultures. The mean number of TH-positive cells in the PDGF-BB-treated rat culture was 64% and 106% higher than in the control cultures after 7 and 10 days in vitro, respectively. Corresponding figures for human TH-positive neurons were 90% and 145%. The influence of PDGF-BB was specific for TH-positive neurons and not a general trophic effect, since no change of either total cell number or metabolic activity was found. In PDGF-BB-treated cultures of human but not rat tissue the TH-positive neurons had longer neurites than observed in control or PDGF-AA-treated cultures. These data indicate that PDGF-BB may act as a trophic factor for mesencephalic dopaminergic neurons and suggest that administration of PDGF-BB could ameliorate degeneration and possibly promote axonal sprouting of these neurons in vivo.

Rat brain capillary endothelial cells express functional PDGF B-type receptors

Immunohistochemical staining revealed the presence of platelet-derived growth factor (PDGF) B-type receptors on capillaries of normal rat brain. Furthermore, capillary endothelial cells isolated from rat brain and grown in tissue culture bound [125I]PDGF-BB but not [125I]PDGF-AA, suggesting that they expressed B-type, but not A-type, PDGF receptors. PDGF-BB and PDGF-AB, but not PDGF-AA, also stimulated incorporation of [3H]thymidine into these cells. Thus, rat brain capillary endothelial cells have functional B-type receptors, and thereby differ from endothelial cells derived from large blood vessels, that do not express PDGF receptors. Our data suggest a possible role for PDGF-BB as an angiogenic factor.

Tissue localization of beta receptors for platelet-derived growth factor and platelet-derived growth factor B chain during wound repair in humans.

The expression and localization of PDGF beta receptors and PDGF-AB/BB in human healing wounds was evaluated by immunohistochemical techniques and in situ hybridization. Expression of PDGF beta receptor protein and PDGF-AB/BB were analyzed in wound margin biopsies using the PDGFR-B2 and PDGF 007 antibodies. PDGF beta receptor expression was minor in normal skin. An increased expression of PDGF beta receptor protein was prominent in vessels in the proliferating tissue zone in wounds as early as 1 d after surgery and was apparent < or = 4 wk after surgery. There was also a concordant increase in PDGF beta receptor mRNA detected by in situ hybridization. PDGF-AB/BB was present in healing wounds as well as in normal skin. In normal skin, expression of PDGF-AB/BB was confined to peripheral nerve fibers and to solitary cells of the epidermis and of the superficial dermis. In wounds, infiltrating mononuclear cells also stained for PDGF-AB/BB. To identify cell types expressing PDGF AB/BB and PDGF beta receptors, respectively, we performed double immunofluorescence stainings. PDGF beta receptors were expressed by vascular smooth muscle cells and cells in capillary walls; the receptor protein could not be detected in neurofilament containing structures, T lymphocytes, or CD68 expressing macrophages. PDGF-AB/BB colocalized with neurofilaments, it was present in Langerhans cells of the epidermis and in HLA-DR positive cells located in the epidermal/dermal junction area. Of the macrophages infiltrating the wound, 43 +/- 18% stained positively for PDGF AB/BB. Since PDGF-AB/BB and PDGF beta receptors are expressed in the healing wound, two essential prerequisites for a role of PDGF in wound healing are fulfilled.