Biomedical subjects

Manuela Martins-Green

Publications and source records attributed to Manuela Martins-Green.

15 recordsLinked to original sources

PubMed2007

The effects of second-hand smoke on biological processes important in atherogenesis.

BACKGROUND: Atherosclerosis is the leading cause of death in western societies and cigarette smoke is among the factors that strongly contribute to the development of this disease. The early events in atherogenesis are stimulated on the one hand by cytokines that chemoattract leukocytes and on the other hand by decrease in circulating molecules that protect endothelial cells (ECs) from injury. Here we focus our studies on the effects of "second-hand" smoke on atherogenesis. METHODS: To perform these studies, a smoking system that closely simulates exposure of humans to second-hand smoke was developed and a mouse model system transgenic for human apoB100 was used. These mice have moderate lipid levels that closely mimic human conditions that lead to atherosclerotic plaque formation. RESULTS: "Second-hand" cigarette smoke decreases plasma high density lipoprotein levels in the blood and also decreases the ratios between high density lipoprotein and low density lipoprotein, high density lipoprotein and triglyceride, and high density lipoprotein and total cholesterol. This change in lipid profiles causes not only more lipid accumulation in the aorta but also lipid deposition in many of the smaller vessels of the heart and in hepatocytes. In addition, mice exposed to smoke have increased levels of Monocyte Chemoattractant Protein-1 in circulation and in the heart/aorta tissue, have increased macrophages in the arterial walls, and have decreased levels of adiponectin, an EC-protective protein. Also, cytokine arrays revealed that mice exposed to smoke do not undergo the switch from the pro-inflammatory cytokine profile (that develops when the mice are initially exposed to second-hand smoke) to the adaptive response. Furthermore, triglyceride levels increase significantly in the liver of smoke-exposed mice. CONCLUSION: Long-term exposure to "second-hand" smoke creates a state of permanent inflammation and an imbalance in the lipid profile that leads to lipid accumulation in the liver and in the blood vessels of the heart and aorta. The former potentially can lead to non-alcoholic fatty liver disease and the latter to heart attacks.

Adiponectin↗

PubMed2006

Activation of sterol regulatory element-binding proteins (SREBPs) is critical in IL-8-induced angiogenesis.

Angiogenesis is essential in many physiological and pathological processes and can be stimulated by many different factors. To better understand and to manipulate this process more effectively, it would be beneficial to identify molecules common to the signaling pathways stimulated by different classes of angiogenic factors. Sterol regulatory element-binding proteins (SREBPs) are involved in the metabolism of cholesterol and fatty acids, molecules that are critical in membrane biology, and hence, many of the processes involved in angiogenesis. Here, we show that angiogenic factors of different families, such as basic fibroblast growth factor, thrombin, and interleukin (IL)-8, stimulate SREBP activation, whereas nonangiogenic factors, such as transforming growth factor-beta1, do not. We focused our detailed studies on IL-8 in vitro and in vivo, as this chemokine is also involved in inflammation and hence, has the potential to be critical in inflammation-induced angiogenesis, a process common to many diseases. Using human microvascular endothelial cells, a rabbit skin wound-healing model, and the chorioallantoic membrane assay, we show that IL-8 stimulates the activation of SREBP-1 and -2, and this activation is specific and receptor-mediated. SREBP activation leads to activation of RhoA through 3-hydroxy-3-methylglutaryl CoA reductase. RhoA is a small guanosinetriphosphatase, important in cytoskeletal functions, which in turn, are critical in many of the cellular processes needed for angiogenesis. Given that diverse, angiogenic factors use different cell-surface receptors, identification of this common step in the signal-transduction pathway provides the opportunity for novel approaches for prevention and treatment of diseases involving abnormal angiogenesis.

Cell Movement↗

PubMed2006

A molecular mimic demonstrates that phosphorylated human prolactin is a potent anti-angiogenic hormone.

S179D prolactin (PRL) is an experimentally useful mimic of naturally phosphorylated human prolactin. S179D PRL, but not unmodified PRL, was found to be anti-angiogenic in both the chorioallantoic membrane and corneal assays. Further investigation using human endothelial in vitro models showed reduced cell number, reduced tubule formation in Matrigel, and reduced migration and invasion, as a function of treatment with S179D PRL. Analysis of growth factors in human endothelial cells in response to S179D PRL showed: a decreased expression or release of endogenous PRL, heme-oxygenase-1, basic fibroblast growth factor (bFGF), angiogenin, epidermal growth factor and vascular endothelial growth factor; and an increased expression of inhibitors of matrix metalloproteases. S179D PRL also blocked signaling from bFGF in these cells. We conclude that this molecular mimic of a pituitary hormone is a potent anti-angiogenic protein, partly as a result of its ability to reduce utilization of several well-established endothelial autocrine growth loops, partly by its ability to block signaling from bFGF and partly because of its ability to decrease endothelial migration. These findings suggest that circulating levels of phosphorylated PRL may influence the progression of cancer and, furthermore, that S179D PRL may be a useful anti-angiogenic therapeutic.

Angiogenesis Inhibitors↗

PubMed2004

A new generation organ culture arising from cross-talk between multiple primary human cell types.

The inability to experiment directly on humans strongly constrains biomedical research, creating a great need to develop cultures that mimic human tissues and organs as experimental systems that can be used to directly understand and manipulate biological processes. The advent of availability of primary human cells now makes possible engineering of such organ cultures. Here we report the generation of a human "skin" arguably the simplest human tissue. Beginning with three primary cell types taken from adult tissues, this organ culture develops into a mature tissue containing a stratified epithelium and an interconnected network of mature microvessels, with appropriate matrix molecules and cytokines. Surprisingly, pericytes and monocytes appear adjacent to and within "blood" vessels, respectively. These cultures respond appropriately to stimulators of specific biological processes, providing a vehicle to investigate basic biological processes, such as 1) cell-cell and cell-microenvironment interaction; 2) transdifferentiation of one cell type to another and/or differentiation from stem cells present in adult tissues; and 3) opportunities for genetic manipulation of human tissues to understand function. Moreover, this "skin" can potentially be developed into a tailored "living bandage" for patients with impaired healing and can serve as prototype for the development of other human organ cultures.

Capillaries↗

PubMed2004

cCXCR1 is a receptor for cIL-8 (9E3/cCAF) and its N- and C-terminal peptides and is also activated by hIL-8 (CXCL8).

Chemokines are chemotactic cytokines that play important roles in immune responses and wound healing, as well as in pathological conditions such as chronic inflammation and tumorigenesis. The chemokines and their receptors are highly conserved and maintain similar functions in different species. One noteworthy exception is the chemokine interleukin (IL)8/CXC ligand 8 and its specific receptor CXCR1, which are found in humans but are not found in the traditional model organisms, mice and rats. As a consequence, we are using model organisms other than mice to study the functions of IL-8 and CXCR1, as well as the mechanisms involved in receptor activation by IL-8. Toward this goal, we have isolated and characterized a new receptor that is highly homologous to human (h)CXCR1, which we named chicken (c)CXCR1. To determine whether this receptor is activated by cIL-8 and its N- and C-terminal peptides and whether it responds to hIL-8, we expressed cCXCR1 in NIH3T3 cells, which naturally lack this receptor, and used single-cell Ca(2)(+) imaging to detect increases in intracellular Ca(2)(+) and immunoblot analysis to detect extracellular signal-regulated kinase 1/2 phosphorylation. We show that cIL-8, its N and C peptides, and hIL-8 activate cCXCR1. We further show that cIL-8 and hIL-8 stimulate chemotaxis of chicken embryonic fibroblasts, cells that express cCXCR1, and that this effect is specific for each chemokine and this receptor. These results strongly suggest that cCXCR1 is the ortholog for hCXCR1 and that chickens can be used as an effective model system to study the functions of IL-8, its terminal peptides, and its specific receptor CXCR1.

3T3 Cells↗

PubMed2004

Vascular endothelial growth factor activation of sterol regulatory element binding protein: a potential role in angiogenesis.

By stimulating the migration and proliferation of endothelial cells (ECs), vascular endothelial growth factor (VEGF) is a potent angiogenic factor. However, the molecular mechanism involved in the VEGF-induced angiogenesis remains elusive. We hypothesized that sterol regulatory element binding proteins (SREBPs), transcription factors governing cellular lipid homeostasis, play an important role in regulating angiogenesis in response to VEGF. VEGF activated SREBP1 and SREBP2 in ECs, as demonstrated by the increased SREBPs, their cleavage products, and the upregulation of the targeted genes. VEGF-induced SREBP activation depended on SREBP cleavage-activating protein (SCAP), because knocking down SCAP by RNA interference (RNAi) inhibited SREBP activation in response to VEGF. SREBP activation was also blocked by 25-hydroxycholesterol (25-HC). To verify the functional implication of SREBPs in VEGF-induced angiogenesis, we tested the role of SREBPs in EC migration and proliferation. SCAP RNAi or 25-HC inhibited VEGF-induced pseudopodia extension and migration of ECs. Both treatments inhibited VEGF-induced EC proliferation, with cell growth arrested at the G(0)/G(1) phase and a concomitant decrease of the S phase. Blocking the PI3K-Akt pathway inhibited the VEGF-activated SREBPs, demonstrating that PI3K-Akt regulates SREBPs. Consistent with our in vitro data, SREBP1 was detected in newly developed microvasculatures in a rabbit skin partial-thickness wound-healing model. SREBP inhibition also markedly suppressed VEGF-induced angiogenesis in chick embryos. In summary, this study identifies SREBPs as the key molecules in regulating angiogenesis in response to VEGF.

Animals↗

PubMed2004

Effects of "second-hand" smoke on structure and function of fibroblasts, cells that are critical for tissue repair and remodeling.

BACKGROUND: It is known that "second-hand" cigarette smoke leads to abnormal tissue repair and remodelling but the cellular mechanisms involved in these adverse effects are not well understood. Fibroblasts play a major role in repair and remodelling. They orchestrate these processes by proliferating, migrating, and secreting proteins such as, cytokines, growth factors and extracellular matrix molecules. Therefore, we focus our studies on the effects of "second-hand" cigarette smoke on the structure and function of these cells. RESULTS: We used sidestream whole (SSW) smoke, a major component of "second-hand" smoke, primary embryonic fibroblasts, cells that behave very much like wound fibroblasts, and a variety of cellular and molecular approaches. We show that doses of smoke similar to those found in tissues cause cytoskeletal changes in the fibroblasts that may lead to a decrease in cell migration. In addition, we also show that these levels of cigarette smoke stimulate an increase in cell survival that is reflected in an increase and/or activation of stress/survival proteins such as cIL-8, grp78, PKB/Akt, p53, and p21. We further show that SSW affects the endomembrane system and that this effect is also accomplished by nicotine alone. CONCLUSIONS: Taken together, our results suggest that: (i) SSW may delay wound repair because of the inability of the fibroblasts to migrate into the wounded area, leading to an accumulation of these cells at the edge of the wound, thus preventing the formation of the healing tissue; (ii) the increase in cell survival coupled to the decrease in cell migration can lead to a build-up of connective tissue, thereby causing fibrosis and excess scarring.

Animals↗

PubMed2004

The N- and C-terminal peptides of hIL8/CXCL8 are ligands for hCXCR1 and hCXCR2.

Chemokines are small cytokines that function in immune responses, wound healing, and pathological conditions such as chronic inflammation and tumorigenesis. This multifunctionality has been attributed primarily to ligand interaction with multiple or dimerized receptors. However, multifunctionality could also result from interactions of the receptors with small peptides produced by processing of the chemokines. Chemokine peptides are functional in vivo, but it is not yet known whether they can interact with and activate their receptors. The work presented here examines the interactions between the two forms of human interleukin 8 (hIL-8), and its N- and C-peptides, with the chemokine receptors hCXCR1 and hCXCR2. We used a Tet-on retroviral system to introduce CXCR1 into mouse NIH 3T3 cells (that lack endogenous CXCR1) and monitored activation of this receptor by the ligands by using quantitative Ca2+ imaging and mitogen-activated protein kinase (MAPK) activation. We found that the N and C termini of the chemokine can stimulate the respective CXCR1 to induce intracellular Ca2+ release and MAPK activation independent of the other regions of the molecules. Furthermore, we showed that these peptides can also stimulate chemotaxis of several cell types, including primary human microvascular endothelial cells, and that this function is specific and mediated by hCXCR1 and/or hCXCR2. These findings advance understanding of the multifunctionality exhibited by chemokines, reveal a new mode of functional regulation, and may serve as the basis for therapeutic targeting.

Animals↗

PubMed2003

Angiogenic effects of prostaglandin E2 are mediated by up-regulation of CXCR4 on human microvascular endothelial cells.

Stimulation of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) increases the expression of CXCR4 on endothelial cells, rendering these cells more responsive to stromal-derived factor 1 (SDF-1), an angiogenic CXC chemokine and unique ligand for CXCR4. Here, we show that prostaglandin E2 (PGE2) mediates the effects of bFGF and VEGF in up-regulating CXCR4 expression on human microvascular endothelial cells (HMECs). Forskolin or 3-isobutyl-1-methyl xanthine (IBMX), 2 inducers of adenylate cyclase, markedly enhanced, whereas cyclooxygenase (COX) inhibitors including aspirin, piroxicam, and NS398 markedly inhibited CXCR4 expression on HMECs. Furthermore, the ability of PGE2 to augment in vitro tubular formation in SDF-1alpha containing matrigel was inhibited completely by blocking CXCR4. Treatment of bFGF- or VEGF-stimulated HMECs with COX inhibitors blocked tubular formation by about 50% to 70%. Prostaglandin-induced human endothelial cell organization and subsequent vascularization can be inhibited to a greater extent by a neutralizing antibody to human CXCR4 in severe combined immunodeficient mice. Additionally, VEGF- and bFGF-induced angiogenesis in vivo was also inhibited by about 50% by NS-398 or piroxicam, and this inhibitory effect was accompanied by decreased expression of CXCR4 on murine endothelial cells. Consequently, by inducing CXCR4 expression, prostaglandin accounts for about 50% of the tubular formation in vitro and in vivo angiogenic effects of VEGF and bFGF. Moreover, augmentation of CXCR4 expression by VEGF, bFGF, and PGE2 involves stimulation of transcription factors binding to the Sp1-binding sites within the promoter region of the CXCR4 gene. These findings indicate that PGE2 is a mediator of VEGF- and bFGF-induced CXCR4-dependent neovessel assembly in vivo and show that angiogenic effects of PGE2 require CXCR4 expression.

1-Methyl-3-isobutylxanthine↗

PubMed2003

MAP kinase phosphorylation-dependent activation of Elk-1 leads to activation of the co-activator p300.

CBP/p300 recruitment to enhancer-bound complexes is a key determinant in promoter activation by many transcription factors. We present a novel mechanism of activating such complexes and show that pre-assembled Elk-1-p300 complexes become activated following Elk-1 phosphorylation by changes in Elk-1-p300 interactions rather than recruitment. It is known that Elk-1 binds to promoter in the absence of stimuli. However, it is unclear how activation of Elk-1 by mitogen-acivated protein kinase (MAPK)-mediated phosphorylation leads to targeted gene transactivation. We show that Elk-1 can interact with p300 in vitro and in vivo in the absence of a stimulus through the Elk-1 C-terminus and the p300 N-terminus. Phosphorylation on Ser383 and Ser389 of Elk-1 by MAPK enhances this basal binding but, most importantly, Elk-1 exhibits new interactions with p300. These interaction changes render a strong histone acetyltransferase activity in the Elk-1-associated complex that could play a critical role in chromatin remodeling and gene activation. The pre-assembly mechanism may greatly accelerate transcription activation, which is important in regulation of expression of immediate-early response genes, in particular those involved in stress responses.

Acetyltransferases↗

PubMed2003

Histochemical localization of the PBAN receptor in the pheromone gland of Heliothis peltigera.

The presence of the pyrokinin (PK)/ Pheromone biosynthesis activating neuropeptide (PBAN) receptor in pheromone gland cells of Heliothis peltigera females was demonstrated, and its spatial distribution in the ovipositor was visualized with two photo-affinity biotinilated ligands: BpaPBAN1-33NH(2) and BpaArg(27)-PBAN28-33NH(2). Light microscopy histological studies revealed that the gland is contained within the inter-segmental membrane (ISM) between the 8th and 9th abdominal segments. The gland was found to be composed of a single layer of columnar epithelial cells positioned under the inter-segmental cuticle. Similar epithelial cells were also found in the dorsal and ventral regions of the 9th abdominal segment. All regions containing the glandular cells bound both ligands, indicating presence of the PK/PBAN receptor. The patterns obtained with both ligands were similar, hinting at the possibility that either both ligands bind to the same receptor, or, that if there are two distinct receptors, their spatial distribution throughout the gland is very similar.

Animals↗

PubMed2002

The CXC chemokine cCAF stimulates precocious deposition of ECM molecules by wound fibroblasts, accelerating development of granulation tissue.

BACKGROUND: During wound repair, fibroblasts orchestrate replacement of the provisional matrix formed during clotting with tenascin, cellular fibronectin and collagen III. These, in turn, are critical for migration of endothelial cells, keratinocytes and additional fibroblasts into the wound site. Fibroblasts are also important in the deposition of collagen I during scar formation. The CXC chemokine chicken Chemotactic and Angiogenic Factor (cCAF), is highly expressed by fibroblasts after wounding and during development of the granulation tissue, especially in areas where extracellular matrix (ECM) is abundant. We hypothesized that cCAF stimulates fibroblasts to produce these matrix molecules. RESULTS: Here we show that this chemokine can stimulate precocious deposition of tenascin, fibronectin and collagen I, but not collagen III. Studies in culture and in vivo show that tenascin stimulation can also be achieved by the N-terminal 15 aas of the protein and occurs at the level of gene expression. In contrast, stimulation of fibronectin and collagen I both require the entire molecule and do not involve changes in gene expression. Fibronectin accumulation appears to be linked to tenascin production, and collagen I to decreased MMP-1 levels. In addition, cCAF is chemotactic for fibroblasts and accelerates their migration. CONCLUSIONS: These previously unknown functions for chemokines suggest that cCAF, the chicken orthologue of human IL-8, enhances healing by rapidly chemoattracting fibroblasts into the wound site and stimulating them to produce ECM molecules, leading to precocious development of granulation tissue. This acceleration of the repair process may have important application to healing of impaired wounds.

Angiogenesis Inducing Agents↗

PubMed2002

The cxc chemokine cCAF stimulates differentiation of fibroblasts into myofibroblasts and accelerates wound closure.

Chemokines are small cytokines primarily known for their roles in inflammation. More recently, however, they have been implicated in processes involved in development of the granulation tissue of wounds, but little is known about their functions during this process. Fibroblasts play key roles in this phase of healing: some fibroblasts differentiate into myofibroblasts, alpha-smooth muscle actin (SMA)-producing cells that are important in wound closure and contraction. Here we show that the CXC chemokine chicken chemotactic and angiogenic factor (cCAF) stimulates fibroblasts to produce high levels of alpha-SMA and to contract collagen gels more effectively than do normal fibroblasts, both characteristic properties of myofibroblasts. Specific inhibition of alpha-SMA expression resulted in abrogation of cCAF-induced contraction. Furthermore, application of cCAF to wounds in vivo increases the number of myofibroblasts present in the granulation tissue and accelerates wound closure and contraction. We also show that these effects in culture and in vivo can be achieved by a peptide containing the NH2-terminal 15 amino acids of the cCAF protein and that inhibition of alpha-SMA expression also results in inhibition of N-peptide-induced collagen gel contraction. We propose that chemokines are major contributors for the differentiation of fibroblasts into myofibroblasts during formation of the repair tissue. Because myofibroblasts are important in many pathological conditions, and because chemokines and their receptors are amenable to pharmacological manipulations, chemokine stimulation of myofibroblast differentiation may have implications for modulation of functions of these cells in vivo.

Actins↗

PubMed2002

Combined administration of antibodies to human interleukin 8 and epidermal growth factor receptor results in increased antimetastatic effects on human breast carcinoma xenografts.

PURPOSE: Current antibody-based immunotherapeutic approaches under evaluation for breast carcinoma are limited in target scope. For example, administration of the human epidermal growth factor receptor (EGFR) antibody, alone or in combination with a chemotherapeutic drug, is thought to primarily inhibit tumor cell proliferation. The aim of this study was to assess the effects of a combined blockade designed to inhibit tumor growth by inhibition of proliferation rate and the proinflammatory effects of interleukin (IL) 8. EXPERIMENTAL DESIGN: A human breast carcinoma cell line that produces high levels of IL-8 was injected s.c. into severe combined immunodeficient mice. IL-8 has been reported to augment the progression of some human tumors; thus, we used a human IL-8 antibody, ABXIL8, in combination with anti-EGFR, ABXEGFR, to inhibit the metastasis of MDA231 tumors. RESULTS: Whereas anti-IL-8 alone had no appreciable antimetastatic effect, the combination of ABXIL8 significantly enhanced the antitumor effects of ABXEGFR, resulting in greater survival of SCID tumor-bearing mice. This effect on survival was correlated with decreased metastatic spread and decreased tumor size in mice receiving both antibodies. Intriguingly, in vitro studies indicate that this antibody combination markedly inhibited matrix metalloproteinase activity associated with MDA-231 cells to a greater degree than either antibody alone. CONCLUSION: Combined administration of these two human antibodies using growth factor blockade in conjunction with chemokine blockade may thus provide a more effective approach for treatment of metastatic human breast carcinoma.

Animals↗

PubMedDate not supplied

Firsthand cigarette smoke alters fibroblast migration and survival: implications for impaired healing.

Although it is known that high levels of cigarette smoke lead to cell death, little is known about the effects of low-to-moderate levels of smoke components that are found in vivo, such as those experienced by cells in tissues. Clinical studies and experimental data show that smokers heal poorly and are more prone to develop fibrotic diseases. Here we show the effects of first-hand cigarette smoke on fibroblasts, cells that are critically involved in these processes. Using doses of smoke found in the tissues of smokers and a variety of cell and molecular approaches, we show that these doses of cigarette smoke do not cause cell death but rather stimulate fibroblasts to produce stress response and survival proteins such as interleukin-8, PKB/Akt, p53, and p21 that in turn contribute to an increase in cell survival. In addition, smoke-treated cells show a decrease in cell migration, which can be explained by the increased cell adhesion and alterations in cytoskeletal elements. We also show that these levels of smoke cause changes in mitochondrial morphology with a minimum loss of function and these changes are the result of exposure to reactive oxygen species. We conclude that the increase in cell survival may lead to a build-up of connective tissue in the area of a wound, potentially leading to delayed healing and/or fibrosis and that the alterations in the cytoskeleton and in cell adhesion result in inhibition of cell migration, a process that could lead to nonclosure of the wound for lack of proper fibroblast migration to form the healing tissue.

Cell Movement↗