IntroductionDifferentially polarized macrophages, especially YM1+ and MHCII+ macrophages, play an... more IntroductionDifferentially polarized macrophages, especially YM1+ and MHCII+ macrophages, play an important role in asthma development. The origin of these polarized macrophages has not been elucidated yet. We therefore aimed to investigate how proliferation, monocyte recruitment, and/or switching of polarization states contribute to this specific pool of polarized interstitial and alveolar macrophages during development of house dust mite (HDM)-induced allergic lung inflammation in mice.MethodsMale and female mice were first treated intranasally with PKH26 to label lung-resident macrophages and were then exposed to either HDM or phosphate-buffered saline (PBS) for two weeks. Different myeloid immune cell types were quantified in lung tissue and blood using flow cytometry.ResultsWe found that macrophage polarization only starts up in the second week of HDM exposures. Before this happened, unpolarized alveolar and interstitial macrophages transiently increased in HDM-exposed mice. Th...
Exacerbations of COPD can be triggered by viral infections. Macrophages are involved in airway in... more Exacerbations of COPD can be triggered by viral infections. Macrophages are involved in airway inflammation in COPD and in limiting inflammatory events in the lung. These opposing tasks are handled by differentially polarized macrophages. MHCII-high expressing macrophages are pro-inflammatory and critical in efficient immune responses against intracellular pathogens, whereas macrophages expressing YM1 are important in healing. To gain more insight into antiviral responses of macrophages in COPD, we studied macrophage responses to X31 influenza in mice after cigarette smoke (CS) exposure as a model of COPD exacerbation. Female BALB/c mice were exposed to CS, each day during 4 weeks. After 3 weeks, mice were exposed to influenza X31. Lung macrophage polarization was measured by flow cytometry. Alveolar macrophages (AM) were defined as CD64+CD68+ SiglecF+CD11c+ and interstitial macrophages (IM) as CD64+CD68+SiglecF-CD11c-. Mice exposed to X31 had more MHCII-high expressing AM than any of the other groups, whereas mice exposed to CS+X31 had more YM1-expressing AM than CS or X31 exposure alone. For IM we found more MHCII-high expressing IM in X31-exposed mice, independent of CS-exposure. In addition, CS+X31 co-exposed mice had higher numbers of YM1-expressing IM than any of the other groups. In summary, AM of CS-exposed mice appear unable to upregulate MHCII during infection with X31 as compared to X31-exposure alone. Together with a polarization shift towards YM1-expression this indicates impaired antigen presentation and defense against intracellular pathogens, which may result in impaired antiviral responses. A role for dysfunctional macrophage polarization in the development of COPD exacerbations is therefore indicated.
Platelet Derived Growth Factor (PDGF) plays a key role in the development of fibrotic processes i... more Platelet Derived Growth Factor (PDGF) plays a key role in the development of fibrotic processes in several tissues. Accordingly, the PDGFβ receptor is abundantly present in these fibrotic tissues. Specific targeting to this receptor is established for a series of compounds in different animal models, all sharing the same targeting moiety, i.e. the cyclic peptide pPB. One of those compounds is pPB-HSA, which might function as carrier vehicle that binds to the PDGFβ-receptor without eliciting an intracellular response itself. When formulated as a solution for parenteral administration, targeting of fibrosis with this proteinaceous construct would involve long-term or even lifelong daily injections, which may jeopardize patient compliance. Therefore, the aim of this study was to develop a solid formulation for the sustained release of pPB-HSA and assess the delivery of the intact protein construct in vivo. pPB-HSA was encapsulated in biodegradable polymeric microspheres using a W/O/W e...
Purpose Rho-kinase regulates activation of hepatic stellate cells (HSC) during liver fibrosis, bu... more Purpose Rho-kinase regulates activation of hepatic stellate cells (HSC) during liver fibrosis, but the ubiquitous presence of this kinase may hinder examination of its exact role and the therapeutic use of inhibitors. We therefore coupled the Rhokinase inhibitor Y27632 to a drug carrier that binds the mannose-6-phosphate insulin-like growth factor II (M6P/IGFII)receptor which is upregulated on activated HSC. Methods Y27632 was coupled to mannose-6-phosphate human serum albumin (M6PHSA), and in vitro experiments were performed on primary rat HSC. Biodistribution and effect studies were performed in an acute CCl 4 model in mice. Results Y27-conjugate remained stable in serum, while drug was efficiently released in liver homogenates. Receptorblocking studies revealed that it was specifically taken up through the M6P/IGFII-receptor on fibroblasts, and it inhibited expression of fibrotic markers in activated HSC. In vivo, liver drug levels were significantly higher after injection of Y27conjugate as compared to Y27632, and the conjugate accumulated specifically in HSC. After acute CCl 4-induced liver injury, Y27-conjugate reduced the local activation of HSC, whereas an equimolar dose of free drug did not. Conclusions We conclude that specific targeting of a Rhokinase inhibitor to HSC leads to enhanced accumulation of the drug in HSC, reducing early fibrogenesis in the liver. KEY WORDS drug targeting. insulin-like growth factor II receptor. kinase inhibitor. liver fibrosis. Rho-kinase ABBREVIATIONS α-SMA α-smooth muscle actin HSC Hepatic stellate cells M6PHSA Mannose-6-phosphate human serum albumin M6P/IGFIIreceptor Mannose-6-phosphate/insulin-like growth factor II receptor ULS™ Universal Linkage System
ABSTRACT Background / Purpose: Interferon γ is endowed with potent anti-fibrotic activities but i... more ABSTRACT Background / Purpose: Interferon γ is endowed with potent anti-fibrotic activities but its clinical use is hampered by adverse effects due to the ubiquitous presence of its receptor. Therefore, we coupled the signaling moiety of IFNγ (IFNγ peptidomimetic) to a PDGFβ receptor recognizing peptide, since the PDGFβ receptor is highly expressed on hepatic stellate cells (HSC), the key cells during liver fibrogenesis.Here we aimed to deliver IFNγ peptidomimetic (mimIFNγ) to HSC to study the effects on acute and advanced liver fibrosis. Main conclusion: The peptidomimetic of IFNγ is stable mimetic peptide of IFNγ that retains the biological activities of IFNγ but lacks the extracellular IFNγ receptor site and therefore cannot induce off-target effects via binding to ubiquitously expressed IFNγR. If redirected via a target receptor PDGFγR, which is highly up-regulated on activated HSC, it can mediate its specific action on the designated target cell. Cell-specific delivery of interferon γ peptidomimetic induced substantial attenuation of acute and advanced liver fibrosis in mice. Through this drug delivery approach, we could largely limit the systemic adverse effects which are otherwise elicited by untargeted IFNγ. In addition, strict species restriction of IFNγ can be bypassed by using mimIFNγ, which is species non-specific.This study clearly demonstrates the potential benefits of PDGFγR-mediated delivery of IFNγ peptidomimetic to fibrotic tissue. In future perspective, this drug targeting approach opens new opportunities to develop cell-selective therapies with increased therapeutic efficacy and reduced adverse effects.
72±19% vs. 52±11%; p = 0.040) rats. Protein expression of VEGFR2 was significantly reduced in spl... more 72±19% vs. 52±11%; p = 0.040) rats. Protein expression of VEGFR2 was significantly reduced in splanchnic tissue (p = 0.049) and in the liver (p = 0.006) of BDL animals. No significant differences in splanchnic or hepatic protein expression were observed for VEGF (p = 0.304, p = 0.118), TNFa (p = 0.215, p = 0.164) or PDGFb (p = 0.43, p = 0.18). In BDL rats, splanchnic (p = 0.007) and hepatic (p = 0.048) PlGF mRNA expression was significantly reduced by LENA treatment. In BDL, a reduction of VEGF-mRNA levels in splachnic tissue (p = 0.012) and the liver (p = 0.082) was observed. LENA treatment did not influence TNFa or PDGFb mRNA expression in BDL rats, neither in splanchnic tissue nor in the liver. In PPVL animals splanchnic protein expression of CD31 was significantly decreased (p = 0.003), while TNFa was increased (p = 0.044). Conclusion: Lenalidomide ameliorates portal hypertensive syndrome in cirrhotic and non-cirrhotic portal hypertensive rats by decreasing proinflammatory and antiangiogenic signaling. Therefore, lenalidomide has some potential as a novel therapeutic option in cirrhotic patients with portal hypertension.
Tumor stromal cells significantly contribute to tumor growth and abundantly express platelet-deri... more Tumor stromal cells significantly contribute to tumor growth and abundantly express platelet-derived growth factor-beta receptor (PDGF-βR). In this study, we targeted stromal as well as tumor cells using our PDGF-βR binding carrier (pPB-HSA). pPB-HSA showed PDGF-βR-specific binding in-vitro and, in-vivo it rapidly accumulated in C26 tumors in mice after i.v. injection. We conjugated doxorubicin to pPB-HSA and, the conjugate showed antitumor effects in-vitro in tumor and stromal cells and in-vivo in C26-tumor bearing mice.
Tumor stromal cells have been recently recognized to contribute to tumor growth. Therefore, we hy... more Tumor stromal cells have been recently recognized to contribute to tumor growth. Therefore, we hypothesized that delivery of anticancer drugs to these cells in addition to the tumor cells might treat cancer more effectively. Stromal cells abundantly expressed Platelet-Derived Growth Factor Receptor-beta (PDGFR-β) in different human tumors as shown with immunohistochemistry. To achieve targeting through PDGFR-β, we developed a carrier by modifying albumin with a PDGFR-β recognizing cyclic peptide (pPB-HSA). pPB-HSA specifically bound to PDGFR-β-expressing 3T3 fibroblasts, C26 and A2780 cancer cells in vitro. Subsequently, doxorubicin was conjugated to pPB-HSA through an acid-sensitive hydrazone linkage. In vitro, Dox-HSA-pPB was taken up by fibroblasts and tumor cells and a short exposure of the conjugate induced cell death in these cells. In vivo, the conjugate rapidly accumulated into PDGFR-β expressing cells in C26 tumors. Treatment with Dox-HSA-pPB significantly reduced the C26 tumor growth in mice while free doxorubicin treated mice had lower response to the therapy. Furthermore, in contrast to free doxorubicin the conjugate did not induce loss in body weight. In conclusion, the present study reveals a novel approach to target key cell types in tumors through PDGFR-β, which can be applied to enhance the therapeutic efficacy of anticancer drugs.
Interferon gamma (IFNγ) is a potent cytokine that displays a variety of anti-viral, anti-prolifer... more Interferon gamma (IFNγ) is a potent cytokine that displays a variety of anti-viral, anti-proliferative, immunomodulatory, apoptotic and anti-fibrotic functions. However, its clinical use is limited to the treatment of few diseases due to the rapid clearance from the body. PEGylated IFN-alpha formulations are shown to be beneficial in viral hepatitis, but PEGylation of IFNγ to enhance its therapeutic effects in liver fibrosis is not yet explored. Liver fibrosis is characterized by the extensive accumulation of an abnormal extracellular matrix and is the major cause of liver-related morbidity and mortality worldwide. To date, there is no pharmacotherapy available for this disease. We modified IFNγ with different-sized linear PEG molecules (5, 10 and 20 kDa) and assessed the biological activity in vitro and in vivo. All PEGylated IFNγ constructs were biologically active and activated IFNγ signaling in vitro as determined with a nitric oxide release assay and a pGAS-Luc reporter plasmid assay, respectively. Similar to IFNγ, all PEGylated IFNγ induced a significant reduction of fibrotic parameters in mouse NIH3T3 fibroblasts as shown with immunohistochemical staining and quantitative PCR analyses. In vivo, the pharmacokinetic profile of radiolabeled 125 I-IFNγ-PEG conjugates revealed a decreased renal clearance and an increased plasma half-life with an increase of PEG size. Moreover, the liver accumulation of PEGylated IFNγ constructs was significantly higher than the unmodified IFNγ, which was also confirmed by increased MHC-II expression in the livers. Furthermore, in a CCl 4-induced acute liver injury model in mice, PEGylated constructs reduced the early fibrotic parameters more drastically than unmodified IFNγ. Of note, these effects were stronger with higher PEG-sized IFNγ constructs. These data nicely correlated with the pharmacokinetic data. In conclusion, PEGylation significantly improved the pharmacokinetics, liver uptake and anti-fibrotic effects of IFNγ. This study opens new opportunities to exploit the therapeutic applications of PEGylated IFNγ for the treatment of liver fibrosis and other diseases.
IntroductionDifferentially polarized macrophages, especially YM1+ and MHCII+ macrophages, play an... more IntroductionDifferentially polarized macrophages, especially YM1+ and MHCII+ macrophages, play an important role in asthma development. The origin of these polarized macrophages has not been elucidated yet. We therefore aimed to investigate how proliferation, monocyte recruitment, and/or switching of polarization states contribute to this specific pool of polarized interstitial and alveolar macrophages during development of house dust mite (HDM)-induced allergic lung inflammation in mice.MethodsMale and female mice were first treated intranasally with PKH26 to label lung-resident macrophages and were then exposed to either HDM or phosphate-buffered saline (PBS) for two weeks. Different myeloid immune cell types were quantified in lung tissue and blood using flow cytometry.ResultsWe found that macrophage polarization only starts up in the second week of HDM exposures. Before this happened, unpolarized alveolar and interstitial macrophages transiently increased in HDM-exposed mice. Th...
Exacerbations of COPD can be triggered by viral infections. Macrophages are involved in airway in... more Exacerbations of COPD can be triggered by viral infections. Macrophages are involved in airway inflammation in COPD and in limiting inflammatory events in the lung. These opposing tasks are handled by differentially polarized macrophages. MHCII-high expressing macrophages are pro-inflammatory and critical in efficient immune responses against intracellular pathogens, whereas macrophages expressing YM1 are important in healing. To gain more insight into antiviral responses of macrophages in COPD, we studied macrophage responses to X31 influenza in mice after cigarette smoke (CS) exposure as a model of COPD exacerbation. Female BALB/c mice were exposed to CS, each day during 4 weeks. After 3 weeks, mice were exposed to influenza X31. Lung macrophage polarization was measured by flow cytometry. Alveolar macrophages (AM) were defined as CD64+CD68+ SiglecF+CD11c+ and interstitial macrophages (IM) as CD64+CD68+SiglecF-CD11c-. Mice exposed to X31 had more MHCII-high expressing AM than any of the other groups, whereas mice exposed to CS+X31 had more YM1-expressing AM than CS or X31 exposure alone. For IM we found more MHCII-high expressing IM in X31-exposed mice, independent of CS-exposure. In addition, CS+X31 co-exposed mice had higher numbers of YM1-expressing IM than any of the other groups. In summary, AM of CS-exposed mice appear unable to upregulate MHCII during infection with X31 as compared to X31-exposure alone. Together with a polarization shift towards YM1-expression this indicates impaired antigen presentation and defense against intracellular pathogens, which may result in impaired antiviral responses. A role for dysfunctional macrophage polarization in the development of COPD exacerbations is therefore indicated.
Platelet Derived Growth Factor (PDGF) plays a key role in the development of fibrotic processes i... more Platelet Derived Growth Factor (PDGF) plays a key role in the development of fibrotic processes in several tissues. Accordingly, the PDGFβ receptor is abundantly present in these fibrotic tissues. Specific targeting to this receptor is established for a series of compounds in different animal models, all sharing the same targeting moiety, i.e. the cyclic peptide pPB. One of those compounds is pPB-HSA, which might function as carrier vehicle that binds to the PDGFβ-receptor without eliciting an intracellular response itself. When formulated as a solution for parenteral administration, targeting of fibrosis with this proteinaceous construct would involve long-term or even lifelong daily injections, which may jeopardize patient compliance. Therefore, the aim of this study was to develop a solid formulation for the sustained release of pPB-HSA and assess the delivery of the intact protein construct in vivo. pPB-HSA was encapsulated in biodegradable polymeric microspheres using a W/O/W e...
Purpose Rho-kinase regulates activation of hepatic stellate cells (HSC) during liver fibrosis, bu... more Purpose Rho-kinase regulates activation of hepatic stellate cells (HSC) during liver fibrosis, but the ubiquitous presence of this kinase may hinder examination of its exact role and the therapeutic use of inhibitors. We therefore coupled the Rhokinase inhibitor Y27632 to a drug carrier that binds the mannose-6-phosphate insulin-like growth factor II (M6P/IGFII)receptor which is upregulated on activated HSC. Methods Y27632 was coupled to mannose-6-phosphate human serum albumin (M6PHSA), and in vitro experiments were performed on primary rat HSC. Biodistribution and effect studies were performed in an acute CCl 4 model in mice. Results Y27-conjugate remained stable in serum, while drug was efficiently released in liver homogenates. Receptorblocking studies revealed that it was specifically taken up through the M6P/IGFII-receptor on fibroblasts, and it inhibited expression of fibrotic markers in activated HSC. In vivo, liver drug levels were significantly higher after injection of Y27conjugate as compared to Y27632, and the conjugate accumulated specifically in HSC. After acute CCl 4-induced liver injury, Y27-conjugate reduced the local activation of HSC, whereas an equimolar dose of free drug did not. Conclusions We conclude that specific targeting of a Rhokinase inhibitor to HSC leads to enhanced accumulation of the drug in HSC, reducing early fibrogenesis in the liver. KEY WORDS drug targeting. insulin-like growth factor II receptor. kinase inhibitor. liver fibrosis. Rho-kinase ABBREVIATIONS α-SMA α-smooth muscle actin HSC Hepatic stellate cells M6PHSA Mannose-6-phosphate human serum albumin M6P/IGFIIreceptor Mannose-6-phosphate/insulin-like growth factor II receptor ULS™ Universal Linkage System
ABSTRACT Background / Purpose: Interferon γ is endowed with potent anti-fibrotic activities but i... more ABSTRACT Background / Purpose: Interferon γ is endowed with potent anti-fibrotic activities but its clinical use is hampered by adverse effects due to the ubiquitous presence of its receptor. Therefore, we coupled the signaling moiety of IFNγ (IFNγ peptidomimetic) to a PDGFβ receptor recognizing peptide, since the PDGFβ receptor is highly expressed on hepatic stellate cells (HSC), the key cells during liver fibrogenesis.Here we aimed to deliver IFNγ peptidomimetic (mimIFNγ) to HSC to study the effects on acute and advanced liver fibrosis. Main conclusion: The peptidomimetic of IFNγ is stable mimetic peptide of IFNγ that retains the biological activities of IFNγ but lacks the extracellular IFNγ receptor site and therefore cannot induce off-target effects via binding to ubiquitously expressed IFNγR. If redirected via a target receptor PDGFγR, which is highly up-regulated on activated HSC, it can mediate its specific action on the designated target cell. Cell-specific delivery of interferon γ peptidomimetic induced substantial attenuation of acute and advanced liver fibrosis in mice. Through this drug delivery approach, we could largely limit the systemic adverse effects which are otherwise elicited by untargeted IFNγ. In addition, strict species restriction of IFNγ can be bypassed by using mimIFNγ, which is species non-specific.This study clearly demonstrates the potential benefits of PDGFγR-mediated delivery of IFNγ peptidomimetic to fibrotic tissue. In future perspective, this drug targeting approach opens new opportunities to develop cell-selective therapies with increased therapeutic efficacy and reduced adverse effects.
72±19% vs. 52±11%; p = 0.040) rats. Protein expression of VEGFR2 was significantly reduced in spl... more 72±19% vs. 52±11%; p = 0.040) rats. Protein expression of VEGFR2 was significantly reduced in splanchnic tissue (p = 0.049) and in the liver (p = 0.006) of BDL animals. No significant differences in splanchnic or hepatic protein expression were observed for VEGF (p = 0.304, p = 0.118), TNFa (p = 0.215, p = 0.164) or PDGFb (p = 0.43, p = 0.18). In BDL rats, splanchnic (p = 0.007) and hepatic (p = 0.048) PlGF mRNA expression was significantly reduced by LENA treatment. In BDL, a reduction of VEGF-mRNA levels in splachnic tissue (p = 0.012) and the liver (p = 0.082) was observed. LENA treatment did not influence TNFa or PDGFb mRNA expression in BDL rats, neither in splanchnic tissue nor in the liver. In PPVL animals splanchnic protein expression of CD31 was significantly decreased (p = 0.003), while TNFa was increased (p = 0.044). Conclusion: Lenalidomide ameliorates portal hypertensive syndrome in cirrhotic and non-cirrhotic portal hypertensive rats by decreasing proinflammatory and antiangiogenic signaling. Therefore, lenalidomide has some potential as a novel therapeutic option in cirrhotic patients with portal hypertension.
Tumor stromal cells significantly contribute to tumor growth and abundantly express platelet-deri... more Tumor stromal cells significantly contribute to tumor growth and abundantly express platelet-derived growth factor-beta receptor (PDGF-βR). In this study, we targeted stromal as well as tumor cells using our PDGF-βR binding carrier (pPB-HSA). pPB-HSA showed PDGF-βR-specific binding in-vitro and, in-vivo it rapidly accumulated in C26 tumors in mice after i.v. injection. We conjugated doxorubicin to pPB-HSA and, the conjugate showed antitumor effects in-vitro in tumor and stromal cells and in-vivo in C26-tumor bearing mice.
Tumor stromal cells have been recently recognized to contribute to tumor growth. Therefore, we hy... more Tumor stromal cells have been recently recognized to contribute to tumor growth. Therefore, we hypothesized that delivery of anticancer drugs to these cells in addition to the tumor cells might treat cancer more effectively. Stromal cells abundantly expressed Platelet-Derived Growth Factor Receptor-beta (PDGFR-β) in different human tumors as shown with immunohistochemistry. To achieve targeting through PDGFR-β, we developed a carrier by modifying albumin with a PDGFR-β recognizing cyclic peptide (pPB-HSA). pPB-HSA specifically bound to PDGFR-β-expressing 3T3 fibroblasts, C26 and A2780 cancer cells in vitro. Subsequently, doxorubicin was conjugated to pPB-HSA through an acid-sensitive hydrazone linkage. In vitro, Dox-HSA-pPB was taken up by fibroblasts and tumor cells and a short exposure of the conjugate induced cell death in these cells. In vivo, the conjugate rapidly accumulated into PDGFR-β expressing cells in C26 tumors. Treatment with Dox-HSA-pPB significantly reduced the C26 tumor growth in mice while free doxorubicin treated mice had lower response to the therapy. Furthermore, in contrast to free doxorubicin the conjugate did not induce loss in body weight. In conclusion, the present study reveals a novel approach to target key cell types in tumors through PDGFR-β, which can be applied to enhance the therapeutic efficacy of anticancer drugs.
Interferon gamma (IFNγ) is a potent cytokine that displays a variety of anti-viral, anti-prolifer... more Interferon gamma (IFNγ) is a potent cytokine that displays a variety of anti-viral, anti-proliferative, immunomodulatory, apoptotic and anti-fibrotic functions. However, its clinical use is limited to the treatment of few diseases due to the rapid clearance from the body. PEGylated IFN-alpha formulations are shown to be beneficial in viral hepatitis, but PEGylation of IFNγ to enhance its therapeutic effects in liver fibrosis is not yet explored. Liver fibrosis is characterized by the extensive accumulation of an abnormal extracellular matrix and is the major cause of liver-related morbidity and mortality worldwide. To date, there is no pharmacotherapy available for this disease. We modified IFNγ with different-sized linear PEG molecules (5, 10 and 20 kDa) and assessed the biological activity in vitro and in vivo. All PEGylated IFNγ constructs were biologically active and activated IFNγ signaling in vitro as determined with a nitric oxide release assay and a pGAS-Luc reporter plasmid assay, respectively. Similar to IFNγ, all PEGylated IFNγ induced a significant reduction of fibrotic parameters in mouse NIH3T3 fibroblasts as shown with immunohistochemical staining and quantitative PCR analyses. In vivo, the pharmacokinetic profile of radiolabeled 125 I-IFNγ-PEG conjugates revealed a decreased renal clearance and an increased plasma half-life with an increase of PEG size. Moreover, the liver accumulation of PEGylated IFNγ constructs was significantly higher than the unmodified IFNγ, which was also confirmed by increased MHC-II expression in the livers. Furthermore, in a CCl 4-induced acute liver injury model in mice, PEGylated constructs reduced the early fibrotic parameters more drastically than unmodified IFNγ. Of note, these effects were stronger with higher PEG-sized IFNγ constructs. These data nicely correlated with the pharmacokinetic data. In conclusion, PEGylation significantly improved the pharmacokinetics, liver uptake and anti-fibrotic effects of IFNγ. This study opens new opportunities to exploit the therapeutic applications of PEGylated IFNγ for the treatment of liver fibrosis and other diseases.
Uploads
Papers by Eduard Post