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P J Hornsby

Publications and source records attributed to P J Hornsby.

At least 19 recordsLinked to original sources
PubMed2006

Adenovirus-delivered DKK3/WNT4 and steroidogenesis in primary cultures of adrenocortical cells.

The Wnt family molecules Dickkopf-3 (DKK3) and WNT4 are present at higher concentrations in the zona glomerulosa than in the rest of the adrenal cortex. In order to study direct effects of these proteins on adrenocortical cell function, we created adenoviruses encoding human DKK3 and WNT4. When added to cultured human adrenocortical cells, DKK3 inhibited aldosterone and cortisol biosynthesis, either alone or together with cyclic AMP. WNT4 increased steroidogenesis when added alone but decreased it in the presence of cyclic AMP. A control adenovirus encoding GFP had no effect. RNA was prepared from cultured cells and was assayed by real-time PCR. CYP11A1 (cholesterol side-chain cleavage enzyme), HSD3B2 (3beta-hydroxysteroid dehydrogenase type II), CYP17 (17 alpha-hydroxylase), CYP21 (21-hydroxylase) and CYP11B1 (11 beta-hydroxylase) mRNAs were all increased by cyclic AMP, whereas CYP11B2 (aldosterone synthase) was unaffected. DKK3 decreased cyclic AMP-stimulated CYP17. WNT4 increased both CYP17 and CYP21 in the absence of cyclic AMP. Both DKK3 and WNT4 increased the level of CYP11B2. These data show that these Wnt signaling molecules have multiple actions on steroidogenesis in adrenocortical cells, including effects on overall steroidogenesis (aldosterone and cortisol biosynthesis) and distinct effects on steroidogenic enzyme mRNA levels. The co-localization of DKK3 and WNT4 in the glomerulosa and their stimulation of CYP11B2 imply an action on glomerulosa-specific function.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Zonal expression of dickkopf-3 and components of the Wnt signalling pathways in the human adrenal cortex.

The mechanisms underlying the differentiation of the adrenal cortex into zones are unclear. Microarray studies on RNA from microdissected zona reticularis (ZR) and zona fasciculata/zona glomerulosa (ZF/ZG) derived from adult human adrenal glands showed that a gene of the dickkopf family (DKK), DKK3, is differentially expressed in the zones. The Dickkopf proteins are morphogens involved in Wnt signalling. Northern blotting showed higher DKK3 transcript levels in ZF/ZG than ZR samples. In situ hybridization on adult human adrenal gland sections showed that DKK3 expression was much higher in the ZG than in the ZF or ZR. DKK3 expression was also higher in the medulla. We screened for expression of other members of the DKK family and the related Wingless-type mouse mammary tumor virus integration site gene family (WNT), frizzled (FZD), and dishevelled (DVL) gene families. Among dickkopf family members, only DKK3 was expressed at a detectable level in both human and mouse adrenocortical RNA samples. Consistent with previously published data on the effects of Wnt4 gene disruption in the mouse, we found only WNT4 expression within the WNT family in both human and mouse RNA. Northern blotting showed that WNT4 was expressed at a higher level in ZF/ZG cells than in ZR. The higher level of DKK3 and WNT4 expression in ZF/ZG cells was confirmed by real-time PCR. In the frizzled and dishevelled families we found FZD1, FZD2 and DVL3 transcripts in human adrenocortical RNA, and FZD2 and DVL3 in mouse adrenocortical RNA. These data show that a variety of genes of the Wnt signalling pathways are expressed in the adrenal cortex. The zonal distribution of DKK3 expression suggests that it could be involved in zonal differentiation or growth.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Contrasting roles of p57(KIP2) and p21(WAF1/CIP1/SDI1) in transplanted human and bovine adrenocortical cells.

Cell transplantation provides a way to compare the regulation of cell proliferation in the same cell type in cell culture and in a vascularized tissue structure in a host animal. The cyclin-dependent kinase inhibitors p57(KIP2), p21(WAF1/CIP1/SDI1) and p27(KIP1) have been extensively studied in cell culture but their role in growth control in tissues is less well understood. In the present experiments we compared the behavior of cell cycle inhibitors in human and bovine adrenocortical cells in culture and following cell transplantation in scid mice. p57 was expressed in the majority of cells in the intact human adrenal cortex. However, double immunofluorescence showed that cells that are in the cell cycle are p57(-) adrenocortical cells, p57 and p27 levels were not affected by inhibition of growth at high cell density, whereas p21 was higher in dividing than growth-inhibited cells. However, p21 was also high in senescent adrenocortical cells. After transplantation of human adrenocortical cells in scid mice, p57 and p27 were observed in most cells in the transplant tissue. Over time the number of p21(+) cells decreased greatly in human adrenocortical cells, but not in bovine adrenocortical cells. This difference correlated with lower levels of cell division (assessed by Ki-67 or incorporation of bromodeoxyuridine) in the human cells in transplant tissues in comparison to bovine cells. The differences between human and bovine cells were observed both when cells were transplanted beneath the kidney capsule and when cells were injected subcutaneously in collagen gel. We conclude that the behavior of p57, but not p21, is consistent with a role as a physiological mediator of proliferative quiescence in the adrenal cortex. The high level of p21 in dividing adrenocortical cells in culture, and in bovine adrenocortical cells in transplant tissues, may be a response to conflicting positive and negative growth influences. Cells may enter the cell cycle under the influence of a strong positive mitogenic signal, but coexisting negative growth stimuli trigger a p21-dependent block to further progression through the cell cycle. This model suggests that bovine adrenocortical cells respond to positive growth stimuli in transplant tissues but human cells lack this response.

Adolescent↗
PubMed2001

Differentially expressed genes in zona reticularis cells of the human adrenal cortex.

The zona reticularis (ZR) cell in the human adrenal cortex is responsible for the secretion of dehydroepiandrosterone, but its biology, origin, and putative decrease in number during aging are poorly understood. In the present experiments, we investigated to what extent ZR and zona fasciculata (ZF) cells differ in patterns of gene expression. Both cell types were purified by microdissection from adult adrenal cortex specimens. After a brief period in culture, RNA was harvested from the cells and used to prepare radioactively labeled probes following amplification by PCR. Probes were used in hybridizations of arrays of cDNAs on nylon membranes (PCR products or plasmids obtained from an adrenal cDNA library). Analysis of hybridization intensities showed that 17 of the 750 genes studied differed in expression by more than 2-fold. Several genes expressed at higher levels in ZR cells encode components of the major histocompatibility complex or enzymes involved in peroxide metabolism. Members of the tubulin gene family were expressed at higher levels in ZF cells. Differential expression of four of the genes was confirmed by Northern blotting. These differences show that although ZR and ZF cells are similar in gene expression, ZR cells have a gene expression pattern related to the unique biology of this cell type.

Adult↗
PubMed2001

Telomere shortening and decline in replicative potential as a function of donor age in human adrenocortical cells.

Telomere shortening is the cause of replicative senescence of mammalian cells in culture and may be a cause of cellular aging in vivo. Some tissues clearly show telomere shortening during aging in humans, but the relationship between replication history and telomere length is obscured by complex relationships between stem cells and more differentiated cell types. Previous experiments on the adrenal cortex and human adrenocortical cells in culture indicate that the proliferative biology of this tissue is relatively simple; cell division occurs continuously throughout life, without evidence for a distinct stem cell compartment. In this tissue we investigated the relationship between telomere biology and replicative senescence by measuring replicative capacity and telomere length as a function of donor age. Cells cultured from adrenal tissue from donors of different ages showed a strong age-related decline in total replicative capacity, falling from about 50 population doublings for fetal cells to an almost total lack of division in culture for cells from older donors. Telomere restriction fragment (TRF) length was analyzed in the same sets of cells and decreased from a value of about 12 kb in fetal cells to approximately 7 kb in cells from older donors. The latter value is consistent with that in fibroblasts which have reached replicative senescence. Furthermore, there was a good correlation in individual donor samples between TRF length and replicative capacity in culture. To confirm the relationship between telomere length, telomerase, and replicative capacity, we measured telomere length in cells before and after infection with a retrovirus encoding hTERT, the catalytic component of human telomerase. The adult adrenal cortex does not have telomerase activity; cells after transduction with the hTERT retrovirus had high telomerase activity. Whereas control cells underwent a replication-dependent shortening in telomeres during long-term growth in culture, hTERT-modified cells maintained telomere length and are probably immortalized. Symmetric cell division in human adrenocortical cells, occurring slowly over the life span, is associated with progressive telomere shortening and may result in proliferative defects in vivo in old age, which could partly account for the age-related changes in the structure and function of the human adrenal cortex.

Adolescent↗
PubMed2001

The human melanocyte: a model system to study the complexity of cellular aging and transformation in non-fibroblastic cells.

The melanocyte is a neural crest-derived cell that localizes in humans to several organs including the epidermis, eye, inner ear and leptomeninges. In the skin, melanocytes synthesize and transfer melanin pigments to surrounding keratinocytes, leading to skin pigmentation and protection against solar exposure. We have investigated the process of replicative senescence and accompanying irreversible cell cycle arrest, in melanocytes in culture. As was found in other cell types, progressive telomere shortening appears to trigger replicative senescence in normal melanocytes. In addition, senescence is associated with increased binding of the cyclin-dependent kinase inhibitor (CDK-I) p16(INK4a) to CDK4, down-regulation of cyclin E protein levels (and consequent loss of cyclin E/CDK2 activity), underphosphorylation of the retinoblastoma protein RB and subsequent increased levels of E2F4-RB repressive complexes. In contrast to fibroblasts, however, the CDK-Is p21(Waf-1) and p27(Kip-1) are also down-regulated. These changes appear to be important for replicative senescence because they do not occur in melanocytes that overexpress the catalytic subunit of the enzyme telomerase (hTERT), or in melanomas, which are tumors that originate from melanocytes or melanoblasts. In contrast to unmodified melanocytes, hTERT overexpressing (telomerized) melanocytes displayed telomerase activity, stable telomere lengths and an extended replicative life span. However, telomerized melanocytes show changes in cell cycle regulatory proteins, including increased levels of cyclin E, p21(Waf-1) and p27(Kip-1). Cyclin E, p21(Waf-1) and p27(Kip-1) are also elevated in many primary melanomas, whereas p16(INK4a) is mutated or deleted in many invasive and metastatic melanomas. Thus, the molecular mechanisms leading to melanocyte senescence and transformation differ significantly from fibroblasts. This suggests that different cell types may use different strategies to halt the cell cycle in response to telomere attrition and thus prevent replicative immortality.

Cell Cycle Proteins↗
PubMed2001

Telomerase activity in primary cultures of normal adrenocortical cells.

Telomerase activity was measured in isolated cells from bovine and human adrenal cortex, in cells in primary culture, in cells in later passages in culture, and in cells genetically modified by expression of hTERT (human telomerase reverse transcriptase). Telomerase activity in freshly isolated bovine adrenocortical cells and in human adrenal cells from donors of various ages (6-79 years) was very low or undetected. However, primary bovine adrenocortical cell cultures were strongly positive for telomerase activity, and primary human adrenocortical cell cultures were weakly positive. Both cell types proliferate in primary culture but proliferation of bovine cells is much more vigorous. When primary bovine cells were subcultured to make successively secondary and tertiary cultures, telomerase activity declined strongly, and was undetected by the third passage. There was only a slight decrease in growth rate over this period. Levels of the telomerase RNA component did not change with passage number when assessed by semi-quantitative competitive RT-PCR. When both bovine and human cells were infected with a retrovirus encoding hTERT, telomerase activity in the cells was very high. We conclude that in the adrenal cortex, as in some other tissues, TERT expression is regulated and upregulation of telomerase activity is associated with rapid proliferation in primary culture. Telomerase activity is not maintained, and introduction of TERT is required for stable telomerase activity and for immortalization.

Adolescent↗
PubMed2001

Transfection by polyethyleneimine-coated microspheres.

UNLABELLED: Polyethyleneimine (PEI) can be used as a DNA delivery mechanism in cell culture and in vivo. Cells can be transfected by using surface-bound PEI, as well as by PEI/DNA microparticles. In the present experiments we extended these observations by preparing microspheres with covalently attached PEI. Blends of poly(epsilon-CBZ-L-lysine) mixed with poly(D,L-lactic-co-glycolic acid) were formed into microspheres using a double-emulsification/solvent evaporation procedure. CBZ (carbobenzoxy) groups on the surface of microspheres were removed by Li(0) /liquid ammonia reduction. Surface amino groups were used for covalent attachment of PEI and other molecules. Silica microspheres with bonded-phase PEI were also used. Microspheres were mixed with plasmid DNA encoding green fluorescent protein and added to cultured cells. PEI-coated microspheres transfected cultured Caco cells and MH-S alveolar macrophages. Expression of the transfected DNA increased over several days. MH-S cells phagocytosed PEI-coated silica microspheres, which were shown to reside in an acidic subcellular compartment. This was demonstrated by conjugating a pH-sensitive fluorescent dye (seminaphthofluorescein, SNAFL) to the microsphere surface. Transfection of MH-S cells was increased when plasmid DNA was complexed with histone on the surface of the microspheres. CONCLUSION: PEI-coated microspheres have potential as a DNA delivery device with advantages of the unique properties of PEI and ease of surface chemical modification.

3T3 Cells↗
PubMed2000

Cutaneous window for in vivo observations of organs and angiogenesis.

BACKGROUND AND OBJECTIVE: The continuous observation in experimental animals of internal organs and processes, such as wound healing and angiogenesis, has been achieved using a variety of transparent windows and chambers. Our objective was to develop procedures for these observations using disposable material for the window and simple surgical techniques. METHODS: For observation of wound healing in the mouse kidney, the kidney was externalized and a wedge was excised. An oval window of polyvinyl chloride film was sutured in place in the skin over the wound. The progress of healing of the wound was observed through the window over 10 days. For observation of angiogenesis, adrenocortical cells were transplanted beneath fascia and muscle and a window was sewn into the skin above the site of transplantation. RESULTS: Clear observations could be made using these cutaneous windows over the period of the experiments. Healing of a wound in the kidney was photographed and measured. The growth of new blood vessels over the site of adrenocortical cell transplantation was observed. CONCLUSIONS: Continuous in vivo observations of organs such as the kidney and processes such as angiogenesis can be made in experimental animals using this simple technique.

3T3 Cells↗
PubMed2000

Formation of functional tissue from transplanted adrenocortical cells expressing telomerase reverse transcriptase.

We report the first use of human telomerase reverse transcriptase (hTERT) expression in experimental xenotransplantation. Previously, we showed that bovine adrenocortical cells can be transplanted into severe combined immunodeficient (SCID) mice, and that these cells form functional tissue that replaces the animals' own adrenal glands. We cotransfected primary bovine adrenocortical cells with plasmids encoding hTERT, SV40 T antigen, neo, and green fluorescent protein. These clones do not undergo loss of telomeric DNA and appear to be immortalized. Two clones were transplanted beneath the kidney capsule of SCID mice. Animals that received cell transplants survived indefinitely despite adrenalectomy. The mouse glucocorticoid, corticosterone, was replaced by the bovine glucocorticoid, cortisol, in the plasma of these animals. The tissue formed from the transplanted cells resembled that formed by transplantation of cells that were not genetically modified and was similar to normal bovine adrenal cortex. The proliferation rate in tissues formed from these clones was low and there were no indications of malignant transformation.

Adrenal Cortex↗
PubMed2000

Transplantation of normal and genetically modified adrenocortical cells.

Cell transplantation techniques have been applied to the study of the biology of the adrenal cortex and to adrenocortical cell proliferation, differentiation, and senescence. Primary bovine adrenocortical cells, primary human adrenocortical cells and genetically modified bovine adrenocortical cells have been transplanted. Successful methods include transplantation of cells beneath the kidney capsule and several subcutaneous cell transplantation procedures. In successful transplants the cells form a functional vascularized tissue structure that allows the host animals to survive adrenalectomy. We show here that subcutaneous cell transplantation does not depend on embedding cells in collagen gel before introduction into the host animal. Subcutaneous transplants secrete both cortisol and aldosterone. However, the variability of plasma aldosterone levels indicates that the factors that determine glomerulosa-type and fasciculata-type cell function in transplant tissues are not well understood.

3T3 Cells↗
PubMed1999

Transplantation of primary bovine adrenocortical cells into scid mice.

Bovine adrenocortical cells were transplanted into scid mice, using a small cylinder inserted beneath the kidney capsule. The tissue formed from primary bovine adrenocortical cells replaced the essential functions of the animals' own adrenal glands, which were removed during the cell transplantation procedure. Most adrenalectomized animals bearing transplanted cells survived indefinitely, whereas adrenalectomized control animals died following surgery. Formation of well-vascularized tissue at the site of transplantation was associated with stable levels of cortisol in the blood, replacing the mouse glucocorticoid (corticosterone). Ultrastructurally, the cultured cells before transplantation had characteristics of rapidly growing cells, but tissue formed in vivo showed features associated with active steroidogenesis. We investigated two potentially critical aspects of the procedure: the provision of support for angiogenesis in the transplant by the inclusion of FGF-secreting 3T3 cells with the adrenocortical cells; and the administration of synthetic steroids as a temporary replacement for steroids lost by adrenalectomy. We found that FGF was required for the rapid formation of well-vascularized tissue, whereas steroid administration avoided some early mortality but was not absolutely required. In contrast to transplants formed from clonal cells, which did not usually secrete aldosterone, transplants formed from primary bovine adrenocortical cells, even though derived from the zona fasciculata, secreted aldosterone as well as cortisol.

3T3 Cells↗
PubMed1999

DNA damage and p21(WAF1/CIP1/SDI1) in experimental injury of the rat adrenal cortex and trauma-associated damage of the human adrenal cortex.

In vivo models are needed to study the reactions of tissues to DNA damage, such as the induction of the cyclin-dependent kinase inhibitor p21, indicating potential repair of the damage, versus apoptosis, indicating the elimination of the damaged cells. Damage to DNA occurs in tissues during shock, sepsis, and other critical medical conditions. Previous studies have found evidence of damage to the cortex of adrenal glands from organ donors who had undergone severe trauma prior to death. The present experiment studied rats under experimental interventions of clinical relevance to patients with conditions that put them at risk for damage to the adrenal glands. These interventions comprised ischaemia and reperfusion injury, sepsis following caecal ligation and puncture, acute pancreatitis, and administration of chemical agents (zymosan and acrylonitrile). All the interventions caused an increase in p21 mRNA as assessed by northern blotting and in situ hybridization. Increased nuclear p21 protein was shown by immunohistochemistry. All the interventions caused damage to DNA, as shown by labelling of available 3' termini of single-strand breaks with terminal transferase. The number of cells undergoing apoptosis, visualized by ligation of a hairpin oligonucleotide probe to double-strand breaks in DNA, was much lower. In rat adrenal glands, apoptotic cells were infrequent under all the conditions studied. They were more abundant in human organ donor adrenal glands that were previously shown to have extensive DNA damage accompanied by induction of p21. The similarity of the effects of a wide variety of surgical interventions and chemical agents suggest a common pathophysiological mechanism which is not specific to the initiating injury. Experimental injury of the rat adrenal cortex provides a model for investigating the role of organ DNA damage and of mediators of the response to DNA damage, such as p21.

Adrenal Cortex↗
PubMed1999

Early events in the formation of a tissue structure from dispersed bovine adrenocortical cells following transplantation into scid mice.

The early events that follow the transplantation of dispersed bovine adrenocortical cells into scid mice were investigated. We introduced adrenocortical cells into a small cylinder inserted beneath the kidney capsule, where they form a tissue structure that becomes vascularized and secretes steroids that replace those from the animal's own adrenal glands, which are removed during the transplantation surgery. We studied cell proliferation, cell survival, apoptosis, and the role of p21WAF1/CIP1/SDI1 over the first 6 days following transplantation. Additionally we examined the invasion of the tissue by host macrophages and endothelial cells. The data show that there is healthy survival of most of the transplanted cells, and that this is related to their position in the cell transplantation cylinder. In the layer of cells that was adjacent to the kidney parenchyma there was a higher rate of cell proliferation and a lower rate of apoptosis than in cells that were located in the upper part of the cylinder. In the lower layer cells were more likely to have nuclear p21, and macrophages and endothelial cells were observed only in this region. Cells that incorporated bromodeoxyuridine administered to animals 2 or 4 days following transplantation were not more likely than other cells to be undergoing a second division when the animals were killed at 6 days, suggesting that proliferation in the lower layer is not confined to a small subpopulation of cells. Among different animals, the extent to which the spaces between the transplanted cells became lined by host endothelial cells was correlated with higher levels of proliferation and nuclear p21, suggesting that vascularization is the critical step for the continued survival and proliferation of the transplant. The present experiments show that bovine adrenocortical cells transplanted into scid mice form a useful model for the study of tissue formation from dispersed cells and the interaction of the transplanted cells with the host.

Adrenal Cortex↗
PubMed1998

Pathophysiologically relevant concentrations of tumor necrosis factor-alpha promote progressive left ventricular dysfunction and remodeling in rats.

BACKGROUND: Although patients with heart failure express elevated circulating levels of tumor necrosis factor-alpha (TNF-alpha) in their peripheral circulation, the structural and functional effects of circulating levels of pathophysiologically relevant concentrations of TNF-alpha on the heart are not known. METHODS AND RESULTS: Osmotic infusion pumps containing either diluent or TNF-alpha were implanted into the peritoneal cavity of rats. The rate of TNF-alpha infusion was titrated to obtain systemic levels of biologically active TNF-alpha comparable to those reported in patients with heart failure (approximately 80 to 100 U/mL), and the animals were examined serially for 15 days. Two-dimensional echocardiography was used to assess changes in left ventricular (LV) structure (remodeling) and LV function. Video edge detection was used to assess isolated cell mechanics, and standard histological techniques were used to assess changes in the volume composition of LV cardiac myocytes and the extracellular matrix. The reversibility of cytokine-induced effects was determined either by removal of the osmotic infusion pumps on day 15 or by treatment of the animals with a soluble TNF-alpha antagonist (TNFR:Fc). The results of this study show that a continuous infusion of TNF-alpha led to a time-dependent depression in LV function, cardiac myocyte shortening, and LV dilation that were at least partially reversible by removal of the osmotic infusion pumps or treatment of the animals with TNFR:Fc. CONCLUSIONS: These studies suggest that pathophysiologically relevant concentrations of TNF-alpha are sufficient to mimic certain aspects of the phenotype observed in experimental and clinical models of heart failure.

Animals↗
PubMed1998

Modification of materials formed from poly(L-lactic acid) to enable covalent binding of biopolymers: application to high-density three-dimensional cell culture in foams with attached collagen.

We describe a method for increasing the hydrophilicity of materials formed from biodegradable polymers and introducing chemical functional groups on their surfaces. Poly(L-lactic acid) was blended with poly(epsilon-CBZ-L-lysine) at an 80:20 ratio. Films of the mixture were prepared and foams were made by solvent casting and salt leaching. Amino groups on the surface of the polymer mixture were deprotected by acid hydrolysis. As an example of the applicability of the technique for attachment of biomolecules, we covalently linked collagen to the deprotected amino groups, creating a surface capable of high density growth of a differentiated cell type (bovine adrenocortical cells). The method should be generally useful for surface modification of biodegradable polymer materials used in tissue engineering.

Adrenal Cortex↗