Acceleration of full-thickness wound healing in normal rats by the synthetic thrombin peptide, TP508.

Thrombin is an essential factor in hemostasis, inflammation, and tissue repair. The synthetic thrombin peptide, TP508, binds to high-affinity thrombin receptors and mimics cellular effects of thrombin at sites of tissue injury. Treatment of full-thickness excisional wounds in normal rats with a single topical application of 0.1 microg TP508 (14 pmol/cm2) reproducibly accelerates wound closure, yielding wounds that on average close 39% more than controls by day 7 (p < 0.001). Wounds treated with 1.0 microg TP508 are 35% and 43% (p < 0.001) smaller than controls on day 7 and 10, respectively. The early rate of closure is approximately 40% greater in TP508-treated than vehicle-treated wounds (20 versus 14 mm2/day) and remains higher through day 7. Breaking strength after closure is slightly greater (15-23%) in wounds treated with TP508 than with saline alone. Histologic comparisons show that TP508 enhances recruitment of inflammatory cells to the wound site within 24 hours post-injury. TP508 treatment also augments revascularization of injured tissue, as evidenced at day 7 by the larger size of functional vessels in the granulation tissue and by the directed development of blood vessels to wounds. These studies raise the possibility that TP508 may be clinically useful in management of open wounds.

Perimembrane localization of the estrogen receptor alpha protein in neuronal processes of cultured hippocampal neurons.

There is clear evidence of rapid, nongenomic responses to estrogen in a variety of neuronal model systems. To address the question of whether some of these rapid estrogen signals might be transduced by the classical estrogen receptor (ER) alpha or a closely related protein in nontransformed neurons, we undertook the present study using isolated fetal rat hippocampal neurons. Several antibodies developed to detect ERalpha were tested in this system and showed positive membrane staining in nonpermeabilized neurons. MC-20, an affinity purified anti-ERalpha, rabbit polyclonal IgG antibody which does not recognize ERbeta was selected to carry out the majority of the experiments. When permeabilized, the hippocampal neurons exhibited low levels of nuclear staining for ERalpha, but abundant labeling for ERalpha throughout the entire cell including the neurites. In addition to traditional immunocytochemistry controls, incubation of neurons for 24 h in the presence of 10 microM antisense oligonucleotide directed against the translation start site of ERalpha reduced ERalpha immunoreactivity throughout the neurons providing further evidence that the immunostaining was specific for ERalpha. Confocal and conventional microscopy demonstrated that the antigen was predominately extranuclear and localization of ERalpha in the neurites suggests that the receptor is in close proximity to the plasma membrane. This localization is consistent with a role for ERalpha as a transducer of rapid, nongenomic estrogen responses in hippocampal neurons.

Antibodies to the estrogen receptor-alpha modulate rapid prolactin release from rat pituitary tumor cells through plasma membrane estrogen receptors.

Antibodies (Abs) raised against the estrogen receptor-alpha (ERalpha) were used to investigate the role of ERalpha proteins located at the plasma membrane in mediating the rapid, estrogen-stimulated secretion of prolactin (PRL) from rat pituitary GH(3)/B6/F10 cells. Exposure of the cells to 1 nM 17beta-estradiol (E(2)) significantly increased PRL release after 3 or 6 min. When ERalpha Abs that bind specifically to ERalpha but are too large to diffuse into cells were tested for activity at the cell membrane, Ab R4, targeted to an ERalpha hinge region sequence, increased PRL release in a time- and concentration-dependent fashion. Ab H151, directed against a different hinge region epitope, decreased PRL release and blocked the stimulatory action of E(2). Abs raised against the DNA binding domain (H226) or the carboxyl terminus (C542) were not biologically active. When each Ab was examined for recognition of ERalpha on the cell surface by immunocytochemistry, all except H151 generated immunostaining in aldehyde-fixed cells. In live cells, however, Ab H151 but not Ab R4 blocked the membrane binding of fluorescently tagged E(2)-BSA. Overall, the data indicate that plasma membrane ERalpha proteins mediate estrogen-stimulated PRL release from GH(3)/B6/F10 cells. These results may also convey information about conformationally sensitive areas of the membrane form of ERalpha involved in rapid, nongenomic responses to estrogens.

Thrombin peptide, TP508, stimulates angiogenic responses in animal models of dermal wound healing, in chick chorioallantoic membranes, and in cultured human aortic and microvascular endothelial cells.

The alpha-thrombin peptide, TP508, accelerates the healing of full-thickness wounds in both normal and ischemic skin. In wounds treated with TP508, a pattern of increased vascularization is consistently observed both grossly and microscopically when compared to wounds treated with saline. One possible mechanism by which the peptide accelerates wound healing is by promoting revascularization of granulation tissue at the injured site. To evaluate the angiogenic potential of TP508, the peptide was tested in the chick embryo chorioallantoic membrane (CAM), where it increased the density and size of CAM blood vessels relative to controls. Additionally, TP508 stimulated chemokinesis and chemotaxis in a dose-dependent fashion in cultured human aortic and human microvascular endothelial cells. Taken together, these in vivo and in vitro data support an angiogenic role for TP508 in wound healing. A working model is presented to explain how this 23-amino-acid peptide, which lacks proteolytic activity, is generated during wound healing and contributes to the nonproteolytic functions associated with alpha-thrombin during tissue repair.

Thrombin peptide TP508 accelerates closure of dermal excisions in animal tissue with surgically induced ischemia.

TP508 is a synthetic peptide corresponding to amino acids 508 through 530 of human prothrombin. We previously demonstrated that a single topical application of TP508 stimulates revascularization and healing of acute incisional and excisional wounds in normal, healthy rat skin. To determine if TP508 would enhance wound healing in ischemic skin, we used bipedicle flaps, cranially based flaps, and free grafts to surgically create ischemic regions on the backs of rats. Full-thickness, circular excisions were made within the flaps or grafts and immediately treated with a single application of saline +/- TP508 (0.1 microg/wound). Compared to wound closure in normal skin, ischemic skin wounds exhibited delayed closure, and the length of delay correlated with the degree of surgically induced ischemia. TP508 significantly accelerated closure in both normal and ischemic skin, resulting in closure rates that were increased within the first 7 days of wounding by 30% in normal tissue and bipedicle flaps, 50% in cranially based flaps, and 225% in free grafts. Moreover, in both flap models, TP508 restored the rate of closure to a rate approximating the control rate observed in normal skin. Histological comparisons of wound tissue from normal skin and cranially based flaps showed that ischemia reduced early recruitment of inflammatory cells at day 1 but increased inflammatory cell numbers in wound beds at day 14. TP508 treatment of ischemic flap wounds significantly increased early inflammatory cell recruitment and restored the normal rapid resolution of the inflammatory phase. In addition, at day 7, TP508-treated wounds appeared to have an increased number of large functional blood vessels compared to saline controls. These studies support the potential efficacy of TP508 in treating ischemic wounds in humans.

Estrogen receptor-alpha detected on the plasma membrane of aldehyde-fixed GH3/B6/F10 rat pituitary tumor cells by enzyme-linked immunocytochemistry.

A population of estrogen receptor-alpha (ER alpha) proteins, located at the plasma membrane, is postulated to mediate the rapid, nongenomic responses of GH3/B6/F10 pituitary cells to estrogen. To demonstrate the presence of ER alpha at the plasma membrane and to distinguish this receptor population from that in the nucleus, GH3/B6/F10 cells were first prepared in 2% paraformaldehyde/0.1% glutaraldehyde in PBS (P/G) without detergent, then exposed to one of several antibodies (Abs) raised against nuclear ER alpha. Ab binding was visualized as a fluorescent/chromagenic reaction product catalyzed by avidin-biotin-complexed alkaline phosphatase. With P/G fixation, Abs could only access antigens at the cell surface, as evidenced by the inability of 70K mol wt dextrans to permeate cells and the absence of intracellular staining by Abs to cytoplasmic or nuclear antigens. ER alpha Abs generated membrane, but not nuclear, staining in P/G-fixed cells; nuclear receptor labeling could only be detected in detergent-treated cells. Specificity of staining for ER alpha was confirmed by three approaches: first, treatment with an antisense oligodeoxynucleotide to nuclear ER alpha mRNA reduced immunolabeling of both membrane and nuclear ER alpha; second, labeling by two Abs raised against different ER alpha oligopeptides was neutralized by competing peptide; third, six Abs (ER21, H226, R4, H222, MC20, and C542) that recognize unique epitopes on rodent ER alpha produced immunolabeling, but neither primate-specific ER alpha Ab nor Ab to ER beta caused staining. In addition to demonstrating the plasma membrane ER alpha in GH3/B6/F10 cells, this method should be applicable to other cell types that exhibit nongenomic responses to estrogen or other steroid hormones.

Rapid actions of estrogens in GH3/B6 pituitary tumor cells via a plasma membrane version of estrogen receptor-alpha.

The focus of our work on rapid actions of estrogens has been on the immuno-identification of a membrane version of the estrogen receptor-alpha (mERalpha) and the correlation of the presence of this receptor to the rapid secretion of prolactin in pituitary tumor cells. We demonstrated the mERalpha by both fluorescence and immuno-enzyme-cytochemistry and with both conventional and confocal microscopy in the cell line GH3/B6 and its sublines. Its presence on cells (including recently subcloned ones) is very heterogenous, unlike the nuclear ERalpha, which is present in every cell. An impeded ligand (estradiol covalently linked to BSA) binds to mERalpha and elicits the same response. A total of eight antibodies to ERalpha recognize mERalpha, making it likely that the membrane and nuclear proteins are highly related. Immuno-identification techniques have also been used to identify mERalpha on the MCF-7 human breast cancer cell line. Estradiol at very low concentrations elicits prolactin release from GH3/B6 cells within a few minutes of application. This response is bimodal, with effective concentrations in both the picomolar and nanomolar ranges. Prolactin release is also elicited or inhibited by ERalpha-specific antibodies. The characteristics of mERalpha and the membrane receptor for glucocorticoids have many similarities, suggesting that this mode of subcellular location/function alternative might be used by other members of the gene family.

The presence of functional estrogen receptors in intestinal epithelial cells.

Reproductive and maturational nutritive needs are examples of situations in which alterations in circulating concentrations of estrogens are associated with changes in intestinal epithelial function. However, it is not clear that any of these effects is due to direct interaction of estrogen with intestinal epithelial estrogen receptors (ER). The experiments reported here were designed to determine whether the small intestinal epithelium contains functional ER and might, therefore, be an estrogen-responsive tissue. IEC-6 cells, a non-transformed line of cells isolated from rat small intestinal crypts, were used for many of the experiments, because they provide a pure preparation of crypt epithelial cells. IEC-6 cells were found to exhibit specific saturable binding of estradiol with a Kd of 5 x 10(-10) M and approximately 100 binding sites/cell. Reverse transcriptase-polymerase chain reaction demonstrated that IEC-6 cells as well as epithelial cells from each segment of the rat intestine (duodenum, jejunum, ileum, and colon) contained ER mRNA of the sequence determined from rat uterus. Estradiol was shown to stimulate IEC-6 cell c-fos mRNA content rapidly and transiently in a manner analogous to that which has been previously demonstrated for other estrogen-responsive tissues. These data demonstrate that intestinal epithelial cells contain ER capable of regulating gene transcription and provide the basis for future studies designed to elucidate the role of estrogens in the regulation of intestinal epithelial function and pathophysiology.