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Platelet-derived growth factor (PDGF)-BB-mediated induction of monocyte chemoattractant protein 1 in human astrocytes: implications for HIV-associated neuroinflammation.

Bethel-Brown C, Yao H, Hu G, Buch S - J Neuroinflammation (2012)

Bottom Line: Furthermore, treating astrocytes with the human recombinant PDGF-BB protein significantly increased the production and release of MCP-1 at both the RNA and protein levels.PDGF-BB-mediated release of MCP-1 was critical for increased permeability in an in vitro BBB model as evidenced by blocking antibody assays.Since MCP-1 is linked to disease severity, understanding its modulation by PDGF-BB could aid in understanding the proinflammatory responses in HAND.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198, USA.

ABSTRACT
Chemokine (C-C motif) ligand 2, also known as monocyte chemoattractant protein 1 (MCP-1) is an important factor for the pathogenesis of HIV-associated neurocognitive disorders (HAND). The mechanisms of MCP-1-mediated neuropathogenesis, in part, revolve around its neuroinflammatory role and the recruitment of monocytes into the central nervous system (CNS) via the disrupted blood-brain barrier (BBB). We have previously demonstrated that HIV-1/HIV-1 Tat upregulate platelet-derived growth factor (PDGF)-BB, a known cerebrovascular permeant; subsequently, the present study was aimed at exploring the regulation of MCP-1 by PDGF-BB in astrocytes with implications in HAND. Specifically, the data herein demonstrate that exposure of human astrocytes to HIV-1 LAI elevated PDGF-B and MCP-1 levels. Furthermore, treating astrocytes with the human recombinant PDGF-BB protein significantly increased the production and release of MCP-1 at both the RNA and protein levels. MCP-1 induction was regulated by activation of extracellular-signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinases and phosphatidylinositol 3-kinase (PI3K)/Akt pathways and the downstream transcription factor, nuclear factor κB (NFκB). Chromatin immunoprecipitation (ChIP) assays demonstrated increased binding of NFκB to the human MCP-1 promoter following PDGF-BB exposure. Conditioned media from PDGF-BB-treated astrocytes increased monocyte transmigration through human brain microvascular endothelial cells (HBMECs), an effect that was blocked by STI-571, a tyrosine kinase inhibitor (PDGF receptor (PDGF-R) blocker). PDGF-BB-mediated release of MCP-1 was critical for increased permeability in an in vitro BBB model as evidenced by blocking antibody assays. Since MCP-1 is linked to disease severity, understanding its modulation by PDGF-BB could aid in understanding the proinflammatory responses in HAND. These results suggest that astrocyte activation by PDGF-BB exaggerates monocyte recruitment into the brain via MCP-1 and underscores the critical role astrocytes play in HAND.

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HIV-1 induces platelet-derived growth factor (PDGF)-B and monocyte chemoattractant protein 1 (MCP-1) mRNA and protein expression in astrocytes. (A) HIV-1 LAI induced PDGF-B chain and MCP-1 mRNA expression in human A172 astrocytes. Total RNA isolated from human A172 astrocytes was subjected to real-time reverse-transcriptase polymerase chain reaction (RT-PCR) analysis using primers specific for human PDGF-B, MCP-1 and 18S. (B) HIV-1 LAI induces PDGF-BB protein expression in human A172 astrocytes. Protein lysates isolated from astrocytes exposed to HIV-1 were subjected to western blot analysis and analyzed for expression of PDGF-BB protein. (C) HIV-1 LAI induces MCP-1 mRNA levels. Total RNA isolated from human A172 astrocytes was subjected to RT-PCR analysis using primers specific for human MCP-1 and 18S. (D) HIV-1 LAI induces MCP-1 protein expression in human A172 astrocytes. Supernatants from astrocytes exposed to HIV-1 for 24 h were subjected to MCP-1 enzyme-linked immunosorbent assay (ELISA) analysis. All the data are presented as mean ± SD of three individual experiments. *P <0.05, **P <0.01, ***P <0.001 versus control group; ##P <0.01 versus HIV-1-treated group.
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Figure 2: HIV-1 induces platelet-derived growth factor (PDGF)-B and monocyte chemoattractant protein 1 (MCP-1) mRNA and protein expression in astrocytes. (A) HIV-1 LAI induced PDGF-B chain and MCP-1 mRNA expression in human A172 astrocytes. Total RNA isolated from human A172 astrocytes was subjected to real-time reverse-transcriptase polymerase chain reaction (RT-PCR) analysis using primers specific for human PDGF-B, MCP-1 and 18S. (B) HIV-1 LAI induces PDGF-BB protein expression in human A172 astrocytes. Protein lysates isolated from astrocytes exposed to HIV-1 were subjected to western blot analysis and analyzed for expression of PDGF-BB protein. (C) HIV-1 LAI induces MCP-1 mRNA levels. Total RNA isolated from human A172 astrocytes was subjected to RT-PCR analysis using primers specific for human MCP-1 and 18S. (D) HIV-1 LAI induces MCP-1 protein expression in human A172 astrocytes. Supernatants from astrocytes exposed to HIV-1 for 24 h were subjected to MCP-1 enzyme-linked immunosorbent assay (ELISA) analysis. All the data are presented as mean ± SD of three individual experiments. *P <0.05, **P <0.01, ***P <0.001 versus control group; ##P <0.01 versus HIV-1-treated group.

Mentions: Since astrocytes in the CNS are exposed to HIV-1, we first sought to examine the modulation of PDGF-B and MCP-1 by HIV-1. Purified HIV-1 LAI virus obtained by high-speed ultracentrifugation and resuspended in astrocyte serum-free media was used for these experiments. Serum-starved astrocytes were exposed to purified virus at a MOI of 0.1 for 6 h followed by assessment of RNA levels by real-time RT-PCR. The MOI of HIV-1 LAI used was based upon our previous study [25]. As shown in Figure 2A, HIV-1 LAI significantly upregulated both PDGF-B (1.7-fold) and MCP-1 (twofold) mRNA levels. To confirm whether increased mRNA levels of PDGF-B translated into increased protein, a western blot analysis was performed on lysates of astrocytes exposed to HIV-1 LAI for 24 h. As shown in Figure 2B exposure to HIV-1 LAI also induced upregulation of PDGF-BB protein. Likewise, supernatants from A172 cells treated with HIV LAI were analyzed for MCP-1 levels via ELISA. As shown in Figure 2D, HIV-1 LAI exposure also resulted in increased MCP-1 levels. To determine whether HIV-mediated induction of MCP-1 could, in part, be explained due to increased PDGF-BB levels, astrocytes were treated with the PDGF receptor blocker STI-571 for 1 h prior to HIV-1 exposure and assessed for MCP-1 expression. Blocking the PDGF-R significantly reduced HIV-mediated induction of MCP-1 RNA (Figure 2C) and protein (Figure 2D).


Platelet-derived growth factor (PDGF)-BB-mediated induction of monocyte chemoattractant protein 1 in human astrocytes: implications for HIV-associated neuroinflammation.

Bethel-Brown C, Yao H, Hu G, Buch S - J Neuroinflammation (2012)

HIV-1 induces platelet-derived growth factor (PDGF)-B and monocyte chemoattractant protein 1 (MCP-1) mRNA and protein expression in astrocytes. (A) HIV-1 LAI induced PDGF-B chain and MCP-1 mRNA expression in human A172 astrocytes. Total RNA isolated from human A172 astrocytes was subjected to real-time reverse-transcriptase polymerase chain reaction (RT-PCR) analysis using primers specific for human PDGF-B, MCP-1 and 18S. (B) HIV-1 LAI induces PDGF-BB protein expression in human A172 astrocytes. Protein lysates isolated from astrocytes exposed to HIV-1 were subjected to western blot analysis and analyzed for expression of PDGF-BB protein. (C) HIV-1 LAI induces MCP-1 mRNA levels. Total RNA isolated from human A172 astrocytes was subjected to RT-PCR analysis using primers specific for human MCP-1 and 18S. (D) HIV-1 LAI induces MCP-1 protein expression in human A172 astrocytes. Supernatants from astrocytes exposed to HIV-1 for 24 h were subjected to MCP-1 enzyme-linked immunosorbent assay (ELISA) analysis. All the data are presented as mean ± SD of three individual experiments. *P <0.05, **P <0.01, ***P <0.001 versus control group; ##P <0.01 versus HIV-1-treated group.
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Figure 2: HIV-1 induces platelet-derived growth factor (PDGF)-B and monocyte chemoattractant protein 1 (MCP-1) mRNA and protein expression in astrocytes. (A) HIV-1 LAI induced PDGF-B chain and MCP-1 mRNA expression in human A172 astrocytes. Total RNA isolated from human A172 astrocytes was subjected to real-time reverse-transcriptase polymerase chain reaction (RT-PCR) analysis using primers specific for human PDGF-B, MCP-1 and 18S. (B) HIV-1 LAI induces PDGF-BB protein expression in human A172 astrocytes. Protein lysates isolated from astrocytes exposed to HIV-1 were subjected to western blot analysis and analyzed for expression of PDGF-BB protein. (C) HIV-1 LAI induces MCP-1 mRNA levels. Total RNA isolated from human A172 astrocytes was subjected to RT-PCR analysis using primers specific for human MCP-1 and 18S. (D) HIV-1 LAI induces MCP-1 protein expression in human A172 astrocytes. Supernatants from astrocytes exposed to HIV-1 for 24 h were subjected to MCP-1 enzyme-linked immunosorbent assay (ELISA) analysis. All the data are presented as mean ± SD of three individual experiments. *P <0.05, **P <0.01, ***P <0.001 versus control group; ##P <0.01 versus HIV-1-treated group.
Mentions: Since astrocytes in the CNS are exposed to HIV-1, we first sought to examine the modulation of PDGF-B and MCP-1 by HIV-1. Purified HIV-1 LAI virus obtained by high-speed ultracentrifugation and resuspended in astrocyte serum-free media was used for these experiments. Serum-starved astrocytes were exposed to purified virus at a MOI of 0.1 for 6 h followed by assessment of RNA levels by real-time RT-PCR. The MOI of HIV-1 LAI used was based upon our previous study [25]. As shown in Figure 2A, HIV-1 LAI significantly upregulated both PDGF-B (1.7-fold) and MCP-1 (twofold) mRNA levels. To confirm whether increased mRNA levels of PDGF-B translated into increased protein, a western blot analysis was performed on lysates of astrocytes exposed to HIV-1 LAI for 24 h. As shown in Figure 2B exposure to HIV-1 LAI also induced upregulation of PDGF-BB protein. Likewise, supernatants from A172 cells treated with HIV LAI were analyzed for MCP-1 levels via ELISA. As shown in Figure 2D, HIV-1 LAI exposure also resulted in increased MCP-1 levels. To determine whether HIV-mediated induction of MCP-1 could, in part, be explained due to increased PDGF-BB levels, astrocytes were treated with the PDGF receptor blocker STI-571 for 1 h prior to HIV-1 exposure and assessed for MCP-1 expression. Blocking the PDGF-R significantly reduced HIV-mediated induction of MCP-1 RNA (Figure 2C) and protein (Figure 2D).

Bottom Line: Furthermore, treating astrocytes with the human recombinant PDGF-BB protein significantly increased the production and release of MCP-1 at both the RNA and protein levels.PDGF-BB-mediated release of MCP-1 was critical for increased permeability in an in vitro BBB model as evidenced by blocking antibody assays.Since MCP-1 is linked to disease severity, understanding its modulation by PDGF-BB could aid in understanding the proinflammatory responses in HAND.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198, USA.

ABSTRACT
Chemokine (C-C motif) ligand 2, also known as monocyte chemoattractant protein 1 (MCP-1) is an important factor for the pathogenesis of HIV-associated neurocognitive disorders (HAND). The mechanisms of MCP-1-mediated neuropathogenesis, in part, revolve around its neuroinflammatory role and the recruitment of monocytes into the central nervous system (CNS) via the disrupted blood-brain barrier (BBB). We have previously demonstrated that HIV-1/HIV-1 Tat upregulate platelet-derived growth factor (PDGF)-BB, a known cerebrovascular permeant; subsequently, the present study was aimed at exploring the regulation of MCP-1 by PDGF-BB in astrocytes with implications in HAND. Specifically, the data herein demonstrate that exposure of human astrocytes to HIV-1 LAI elevated PDGF-B and MCP-1 levels. Furthermore, treating astrocytes with the human recombinant PDGF-BB protein significantly increased the production and release of MCP-1 at both the RNA and protein levels. MCP-1 induction was regulated by activation of extracellular-signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinases and phosphatidylinositol 3-kinase (PI3K)/Akt pathways and the downstream transcription factor, nuclear factor κB (NFκB). Chromatin immunoprecipitation (ChIP) assays demonstrated increased binding of NFκB to the human MCP-1 promoter following PDGF-BB exposure. Conditioned media from PDGF-BB-treated astrocytes increased monocyte transmigration through human brain microvascular endothelial cells (HBMECs), an effect that was blocked by STI-571, a tyrosine kinase inhibitor (PDGF receptor (PDGF-R) blocker). PDGF-BB-mediated release of MCP-1 was critical for increased permeability in an in vitro BBB model as evidenced by blocking antibody assays. Since MCP-1 is linked to disease severity, understanding its modulation by PDGF-BB could aid in understanding the proinflammatory responses in HAND. These results suggest that astrocyte activation by PDGF-BB exaggerates monocyte recruitment into the brain via MCP-1 and underscores the critical role astrocytes play in HAND.

Show MeSH
Related in: MedlinePlus