During my PhD program I addressed my attention on the characterization of the alteration on pulmonary homeostasis induced by different kind of perturbations. The study embraces two aspects, an in-vivo and an in-vitro one that at first may appear very different, but both deal with “insults” to the lung to which the tissue or the cells have to react. Concerning the in-vivo study, the insult is represented by the reduction of oxygen (condition termed hypoxia) while, for the in-vitro study, it is represented by the exposition to nanoparticles. For both studies we analyzed the tissue/cells response, from the physiological point of view. The primary function of the lung is to allow the gas diffusion into and out of the blood, function that is optimized when the thickness of the blood-air barrier is minimal. Hypoxia lead to an accumulation of the extracellular interstitial fluid and so an impairment of gas diffusion. Based on the evidence that, within the same lung, the response to a decrease in oxygen is not homogenous, we hypothesized that the local adaptations develop as a consequence of pre-existing tissue features favoring or preventing fluid extravasation and matrix remodeling. In this scenario, we characterized different regions of rat lung in order to identify differences in extracellular matrix (ECM) status, metabolic sensors and endothelial/epithelial mediators (at the air-blood barrier) that might be the players that trigger the tissue local adaptations. Our data confirmed our hypothesis showing that regional differences are present in the lung: in particular, the lower lobe of the right lung presents a reduced expression of proteins involved in tissue remodeling like the matrix metalloproteinase MMP-2 and the growth factor KGF, this last involved in tissue repair mainly through controlling the matrix deposition. The left lung, in particular the ventral region of the upper lobe, shows a decrease of the proteins expression involved in the transduction of chemical (cavoline) and mechanical (MMP-17) signals. Concerning the oxygen sensor PGC-1α, a cofactor of mitochondria biogenesis, and the endothelial capillary control (VEGF), the key factor of neo vascularization, no differences were found in all lung, both for mRNA and protein levels. Before translating the study to the hypoxic model, we need to optimize the working condition. In fact, to provide a picture, as precise as possible, of the profile expression of genes and protein in a given situation is crucial to correctly interpret the data, but often it is not easy. This is also true for hypoxia. In order to ensure that we will be in the condition to analyze the changes due to the real hypoxic response, we investigated how long the modifications persist before the hypoxia-mediated signal goes back to the control value following the recovery to normoxia. We found that in normoxia HIF-2α is the most relevant isoform of HIFs isoform as evidenced by the low level of his main regulator protein PHD3. Upon chronic hypoxia exposure we found that, in accordance with literature, HIF-1α and not HIF-2α, result overexpressed, becoming, in relative terms, the predominant responsible for tissue response and the more sensitive to the O2 reduction. When the hypoxic stimulus ceases, all the HIFs and PHDs mRNAs considered presented a very rapid decay upon reoxygenation (within 15 minutes from the end of hypoxia). Among the genes under the control of HIFs pathway we considered the Endothelial Nitric Oxide Sinthase (eNOS) and the Vascular Endothelial Growth Factor (VEGF) that, with their activity, balance the constriction or dilation of pulmonary vessels and the needs for neo-angiogenesis. We did not find an hypoxia-dependent increase in VEGF mRNA. Regarding eNOS expression levels our results confirm previously data where, upon chronic hypoxia, it is down regulated at transcriptional and post-transcriptional level with a shortening of the eNOS mRNA half-life. In the last two decades, nanotechnology has attracted increasingly interest for the application of nanoparticles (NPs) in pulmonary drug delivery and bioimaging fields. However, despite a wide number of publication on the application of nanotechnology to biomedicine, the issue associated to NPs acute and late toxicity still remains a major unsolved drawback. In particular, only few papers were dedicated to the description of cellular response following NPs exposure. Accordingly, we provided an essential starting point for elucidating the basics of cell response to NPs exposition, analysing, the cellular uptake and intracellular dynamics of Solid Lipid Nanoparticles loaded with Coumarin-6 (c-SLN) and iron oxide nanoparticles (RSF39@L-dopa-TRITC NPs) by alveolar epithelial cell (A549). Our data revealed that both NPs are biocompatible, showing a wide range of concentration where toxicity remains at zero level, and the integrity of the plasma membrane is preserved. The signal associated with c-SLN enters rapidly in the cells and accumulates in the perinuclear region in a cytoskeleton-dependent way since the disruption of actin filaments by Cytochalasin-D prevents the perinuclear accumulation. Lowering the temperature of incubation from 37°C to 4°C decreased the intracellular signal intensity by about the 48% for both free and loaded fluorophore suggesting that the uptake of the model drug loaded on SLN is only partially related to endocytotic pathway. Moreover, the delivery of Coumarin-6 by SLN, is more efficient respect to the one obtained after incubation of the free fluorophore. Our data clearly suggested a macropinocytosis-mediated uptake of iron oxide NPs as demonstrated by the incubation with specific inhibitors of macropinocytosis (e.g. Amiloride, its derivative EIPA and LY294002) along with Nocodazole and Cytochalasin D that interfere with the process of membrane ruffling. Once inside the cells, RSF39@L-dopa-TRITC NPs do not behave as singles but clustered into aggregates and proceeded towards the peri-nuclear region converging presumably in autophagosomes and finally reaching the more acidic compartments of the cells (lysosomes). Interestingly, the trafficking into the cells and the clusterization of NPs-containing vesicles depend on the integrity of the microtubules rather than on actin-myosin organization The identification of the molecular factors responsible for the local adaptation at tissue level during hypoxia is fundamental prevent the onset of pathological scenarios. The thorough understanding of the intracellular dynamics in response to NPs is very useful and allow to further modify particle properties for optimizing their targeted delivery. The combination of these two new knowledge may represent a novel emerging field for the application of nanomedicine, by delivering agents for diagnostic purpose or drugs able to ameliorate the lung region specific response.

(2013). Transient changes in pulmonary homeostasis: cellular and tissue response. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2013).

Transient changes in pulmonary homeostasis: cellular and tissue response

PANARITI, ALICE LUCIA
2013-01-18

Abstract

During my PhD program I addressed my attention on the characterization of the alteration on pulmonary homeostasis induced by different kind of perturbations. The study embraces two aspects, an in-vivo and an in-vitro one that at first may appear very different, but both deal with “insults” to the lung to which the tissue or the cells have to react. Concerning the in-vivo study, the insult is represented by the reduction of oxygen (condition termed hypoxia) while, for the in-vitro study, it is represented by the exposition to nanoparticles. For both studies we analyzed the tissue/cells response, from the physiological point of view. The primary function of the lung is to allow the gas diffusion into and out of the blood, function that is optimized when the thickness of the blood-air barrier is minimal. Hypoxia lead to an accumulation of the extracellular interstitial fluid and so an impairment of gas diffusion. Based on the evidence that, within the same lung, the response to a decrease in oxygen is not homogenous, we hypothesized that the local adaptations develop as a consequence of pre-existing tissue features favoring or preventing fluid extravasation and matrix remodeling. In this scenario, we characterized different regions of rat lung in order to identify differences in extracellular matrix (ECM) status, metabolic sensors and endothelial/epithelial mediators (at the air-blood barrier) that might be the players that trigger the tissue local adaptations. Our data confirmed our hypothesis showing that regional differences are present in the lung: in particular, the lower lobe of the right lung presents a reduced expression of proteins involved in tissue remodeling like the matrix metalloproteinase MMP-2 and the growth factor KGF, this last involved in tissue repair mainly through controlling the matrix deposition. The left lung, in particular the ventral region of the upper lobe, shows a decrease of the proteins expression involved in the transduction of chemical (cavoline) and mechanical (MMP-17) signals. Concerning the oxygen sensor PGC-1α, a cofactor of mitochondria biogenesis, and the endothelial capillary control (VEGF), the key factor of neo vascularization, no differences were found in all lung, both for mRNA and protein levels. Before translating the study to the hypoxic model, we need to optimize the working condition. In fact, to provide a picture, as precise as possible, of the profile expression of genes and protein in a given situation is crucial to correctly interpret the data, but often it is not easy. This is also true for hypoxia. In order to ensure that we will be in the condition to analyze the changes due to the real hypoxic response, we investigated how long the modifications persist before the hypoxia-mediated signal goes back to the control value following the recovery to normoxia. We found that in normoxia HIF-2α is the most relevant isoform of HIFs isoform as evidenced by the low level of his main regulator protein PHD3. Upon chronic hypoxia exposure we found that, in accordance with literature, HIF-1α and not HIF-2α, result overexpressed, becoming, in relative terms, the predominant responsible for tissue response and the more sensitive to the O2 reduction. When the hypoxic stimulus ceases, all the HIFs and PHDs mRNAs considered presented a very rapid decay upon reoxygenation (within 15 minutes from the end of hypoxia). Among the genes under the control of HIFs pathway we considered the Endothelial Nitric Oxide Sinthase (eNOS) and the Vascular Endothelial Growth Factor (VEGF) that, with their activity, balance the constriction or dilation of pulmonary vessels and the needs for neo-angiogenesis. We did not find an hypoxia-dependent increase in VEGF mRNA. Regarding eNOS expression levels our results confirm previously data where, upon chronic hypoxia, it is down regulated at transcriptional and post-transcriptional level with a shortening of the eNOS mRNA half-life. In the last two decades, nanotechnology has attracted increasingly interest for the application of nanoparticles (NPs) in pulmonary drug delivery and bioimaging fields. However, despite a wide number of publication on the application of nanotechnology to biomedicine, the issue associated to NPs acute and late toxicity still remains a major unsolved drawback. In particular, only few papers were dedicated to the description of cellular response following NPs exposure. Accordingly, we provided an essential starting point for elucidating the basics of cell response to NPs exposition, analysing, the cellular uptake and intracellular dynamics of Solid Lipid Nanoparticles loaded with Coumarin-6 (c-SLN) and iron oxide nanoparticles (RSF39@L-dopa-TRITC NPs) by alveolar epithelial cell (A549). Our data revealed that both NPs are biocompatible, showing a wide range of concentration where toxicity remains at zero level, and the integrity of the plasma membrane is preserved. The signal associated with c-SLN enters rapidly in the cells and accumulates in the perinuclear region in a cytoskeleton-dependent way since the disruption of actin filaments by Cytochalasin-D prevents the perinuclear accumulation. Lowering the temperature of incubation from 37°C to 4°C decreased the intracellular signal intensity by about the 48% for both free and loaded fluorophore suggesting that the uptake of the model drug loaded on SLN is only partially related to endocytotic pathway. Moreover, the delivery of Coumarin-6 by SLN, is more efficient respect to the one obtained after incubation of the free fluorophore. Our data clearly suggested a macropinocytosis-mediated uptake of iron oxide NPs as demonstrated by the incubation with specific inhibitors of macropinocytosis (e.g. Amiloride, its derivative EIPA and LY294002) along with Nocodazole and Cytochalasin D that interfere with the process of membrane ruffling. Once inside the cells, RSF39@L-dopa-TRITC NPs do not behave as singles but clustered into aggregates and proceeded towards the peri-nuclear region converging presumably in autophagosomes and finally reaching the more acidic compartments of the cells (lysosomes). Interestingly, the trafficking into the cells and the clusterization of NPs-containing vesicles depend on the integrity of the microtubules rather than on actin-myosin organization The identification of the molecular factors responsible for the local adaptation at tissue level during hypoxia is fundamental prevent the onset of pathological scenarios. The thorough understanding of the intracellular dynamics in response to NPs is very useful and allow to further modify particle properties for optimizing their targeted delivery. The combination of these two new knowledge may represent a novel emerging field for the application of nanomedicine, by delivering agents for diagnostic purpose or drugs able to ameliorate the lung region specific response.
RIVOLTA, ILARIA
MISEROCCHI, GIUSEPPE
hypoxia, extracellular matrix, nanotechnology, nanoparticles, solid lipid nanoparticles (SLN), iron oxide nanoparticles, cellular uptake
BIO/09 - FISIOLOGIA
English
Scuola di Dottorato in Medicina Traslazionale e Molecolare
SCUOLA DI DOTTORATO IN MEDICINA TRASLAZIONALE E MOLECOLARE (DIMET) - 72R
25
2011/2012
(2013). Transient changes in pulmonary homeostasis: cellular and tissue response. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2013).
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/10281/44986
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