Pericytes regulate vascular immune homeostasis in the CNS.

Pericytes regulate the development of organ-specific characteristics of the brain vasculature such as the blood-brain barrier (BBB) and astrocytic end-feet. Whether pericytes are involved in the control of leukocyte trafficking in the adult central nervous system (CNS), a process tightly regulated by CNS vasculature, remains elusive. Using adult pericyte-deficient mice (Pdgfbret/ret ), we show that pericytes limit leukocyte infiltration into the CNS during homeostasis and autoimmune neuroinflammation. The permissiveness of the vasculature toward leukocyte trafficking in Pdgfbret/ret mice inversely correlates with vessel pericyte coverage. Upon induction of experimental autoimmune encephalomyelitis (EAE), pericyte-deficient mice die of severe atypical EAE, which can be reversed with fingolimod, indicating that the mortality is due to the massive influx of immune cells into the brain. Additionally, administration of anti-VCAM-1 and anti-ICAM-1 antibodies reduces leukocyte infiltration and diminishes the severity of atypical EAE symptoms of Pdgfbret/ret mice, indicating that the proinflammatory endothelium due to absence of pericytes facilitates exaggerated neuroinflammation. Furthermore, we show that the presence of myelin peptide-specific peripheral T cells in Pdgfbret/ret ;2D2tg mice leads to the development of spontaneous neurological symptoms paralleled by the massive influx of leukocytes into the brain. These findings indicate that intrinsic changes within brain vasculature can promote the development of a neuroinflammatory disorder.

Platelet-derived growth factor beta is a potent inflammatory driver in paediatric high-grade glioma.

Paediatric high-grade gliomas (HGGs) account for the most brain tumour-related deaths in children and have a median survival of 12-15 months. One promising avenue of research is the development of novel therapies targeting the properties of non-neoplastic cell-types within the tumour such as tumour associated macrophages (TAMs). TAMs are immunosuppressive and promote tumour malignancy in adult HGG; however, in paediatric medulloblastoma, TAMs exhibit anti-tumour properties. Much is known about TAMs in adult HGG, yet little is known about them in the paediatric setting. This raises the question of whether paediatric HGGs possess a distinct constituency of TAMs because of their unique genetic landscapes. Using human paediatric HGG tissue samples and murine models of paediatric HGG, we demonstrate diffuse midline gliomas possess a greater inflammatory gene expression profile compared to hemispheric paediatric HGGs. We also show despite possessing sparse T-cell infiltration, human paediatric HGGs possess high infiltration of IBA1+ TAMs. CD31, PDGFRß, and PDGFB all strongly correlate with IBA1+ TAM infiltration. To investigate the TAM population, we used the RCAS/tv-a system to recapitulate paediatric HGG in newborn immunocompetent mice. Tumours are induced in Nestin-positive brain cells by PDGFA or PDGFB overexpression with Cdkn2a or Tp53 co-mutations. Tumours driven by PDGFB have a significantly lower median survival compared to PDGFA-driven tumours and have increased TAM infiltration. NanoString and quantitative PCR analysis indicates PDGFB-driven tumours have a highly inflammatory microenvironment characterized by high chemokine expression. In vitro bone marrow-derived monocyte and microglial cultures demonstrate bone marrow-derived monocytes are most responsible for the production of inflammatory signals in the tumour microenvironment in response to PDGFB stimulation. Lastly, using knockout mice deficient for individual chemokines, we demonstrate the feasibility of reducing TAM infiltration and prolonging survival in both PDGFA and PDGFB-driven tumours. We identify CCL3 as a potential key chemokine in these processes in both humans and mice. Together, these studies provide evidence for the potent inflammatory effects PDGFB has in paediatric HGGs.

[PDGF-B immunogen preparation and the suppressive effect of anti-PDGF-B ascite antibody on the proliferation of hepG2 cells].

To observe the immunogenicity of hPDGF-B immunogens that were synthesized with the fusional expression vector pET28-Trx and to test the suppressive effect of these specific antibodies induced by both of immunogens on proliferation of human HepG2 hepatoma cells. First, we chose 2 antigenic epitopes hPDGF-BΔ103-118aa and hPDGF-BΔ152-167aa from human PDGF-B and inserted these 2 coding regions into the empty vector plasmid pET28-Trx, separately. Second, mice were immunized with purified recombinant proteins to generate polyclonal antibody. Then we intraperitoneally injected mice bearing hepatoma 22 (H22) tumor cells to prepare antibody ascites. ELISA and Western blot were used to detect the titer and the utility of the antibody, respectively. Finally, HepG2 cells were exposed to PDGF-BB protein or anti-PDGF-B ascite antibody in different dilution concentrations groups and the proliferation of HepG2 cells was quantified by CCK8 assay. As the results, we identified mice that could produce high drop of neutralizing antibodies against hPDGF-B induced by both two recombinant proteins. Two anti-PDGF-B ascite antibodies could markedly inhibit the proliferation of HepG2 cells by blocking the stimulating effect of PDGF-BB protein. Our results suggest that Trx-PDGF-B recombinant protein as immunogen provides a new method for the preparation of PDGF-B vaccine, and also a new idea for the treatment of hepatocellular carcinoma in clinical practice.

A signal amplification probe enhances sensitivity of antibodies and aptamers based Immuno-diagnostic assays.

One major unmet need is improving the sensitivity of immune-diagnostic assays. This is particularly important in the field of biomarker discoveries and monitoring. We have established a novel signal amplification probe system enabling a highly sensitive target detection platform to be used in immuno-assays. The probe consists of a double stranded DNA that can carry a large number of signaling elements such as biotin or fluorescent molecules. The DNA probe anchors to the recognition unit, whether an antibody or an aptamer, by covalent conjugation or by a simple and rapid molecular association process. Binding curves obtained by using the DNA amplification probe are dose dependent and linear over a wide range of antigen concentration. The optimal slopes are characterized by high signals and low background increasing the assay sensitivity and reducing the limit of detection by up to 10-fold compared to biotinylated antibodies commonly used in ELISA systems. When using aptamers in combination with the amplification probe for antigen recognition, the limit of detection is comparable to that obtained by biotinylated antibodies. Biotin labeled aptamers practically cannot be used for detection of low target levels. The DNA amplification probe system enables to expand the range of diagnostic assays including clinical samples and meet research needs.

Plasmablasts With a Mucosal Phenotype Contribute to Plasmacytosis in Systemic Lupus Erythematosus.

OBJECTIVE: To analyze the composition of known plasmacytosis in systemic lupus erythematosus (SLE) to obtain further insight into the nature of underlying mechanisms. METHODS: Plasmablasts from patients with active SLE, patients with inactive/treated SLE, and healthy controls were characterized by flow cytometry, enzyme-linked immunospot assay, and Transwell migration assays and compared to vaccination-induced plasmablasts. Serum cytokine levels were analyzed by Luminex assay, and histologic analysis of kidney biopsy specimens was performed. RESULTS: Circulating plasmablasts in SLE expressed markers of mucosal immune reactions. IgA, CCR10, and ß7 integrin were expressed by 48%, 40%, and 38% of plasmablasts, respectively, with varying coexpression patterns. Consistent with mucosal homing, some SLE plasmablasts migrated toward the mucosal chemokine CCL28 and secreted polymeric IgA. SLE plasmablasts shared phenotypic characteristics with antigen-specific plasmablasts induced by oral, but not parenteral, vaccinations. Autoreactive antibody-secreting cells of the IgG and IgA isotypes were detectable, but only the emergence of phenotypically mucosal plasmablasts was positively associated with serum interleukin-2 and platelet-derived growth factor BB levels. CONCLUSION: Our data suggest that distinct plasmablast differentiation pathways jointly contribute to peripheral plasmacytosis in SLE, i.e., a cytokine-amplified mucosal "steady-state" plasmablast response, and an autoreactive plasmablast response, representing conventional autoimmunity. Our results indicate an overly activated mucosal immune system in patients with SLE, with both immunologic and clinical implications.

Effects of ischemic preconditioning on PDGF-BB expression in the gerbil hippocampal CA1 region following transient cerebral ischemia.

Ischemic preconditioning (IPC) is induced by exposure to brief durations of transient ischemia, which results in ischemic tolerance to a subsequent longer or lethal period of ischemia. In the present study, the effects of IPC (2 min of transient cerebral ischemia) were examined on immunoreactivity of platelet­derived growth factor (PDGF)­BB and on neuroprotection in the gerbil hippocampal CA1 region following lethal transient cerebral ischemia (LTCI; 5 min of transient cerebral ischemia). IPC was subjected to a 2­min sublethal ischemia and a LTCI was given 5­min transient ischemia. The animals in all of the groups were given recovery times of 1, 2 and 5 days and change in PDGF­BB immunoreactivity was examined as was the neuronal damage/death in the hippocampus induced by LTCI. LTCI induced a significant loss of pyramidal neurons in the hippocampal CA1 region 5 days after LTCI, and significantly decreased PDGF­BB immunoreactivity in the CA1 pyramidal neurons from day 1 after LTCI. Conversely, IPC effectively protected the CA1 pyramidal neurons from LTCI and increased PDGF­BB immunoreactivity in the CA1 pyramidal neurons post­LTCI. In conclusion, the results demonstrated that LTCI significantly altered PDGF­BB immunoreactivity in pyramidal neurons in the hippocampal CA1 region, whereas IPC increased the immunoreactivity. These findings indicated that PDGF­BB may be associated with IPC­mediated neuroprotection.

The Deficiency of Indoleamine 2,3-Dioxygenase Aggravates the CCl4-Induced Liver Fibrosis in Mice.

In the present study, we examined the role of indoleamine 2,3-dioxygenase (IDO) in the development of CCl4-induced hepatic fibrosis. The liver fibrosis induced by repetitive administration with CCl4 was aggravated in IDO-KO mice compared to WT mice. In IDO-KO mice treated with CCl4, the number of several inflammatory cells and the expression of pro-inflammatory cytokines increased in the liver. In the results, activated hepatic stellate cells (HSCs) and fibrogenic factors on HSCs increased after repetitive CCl4 administration in IDO-KO mice compared to WT mice. Moreover, the treatment with l-tryptophan aggravated the CCl4-induced hepatic fibrosis in WT mice. Our findings demonstrated that the IDO deficiency enhanced the inflammation in the liver and aggravated liver fibrosis in repetitive CCl4-treated mice.

Augmentation of Autoantibodies by Helicobacter pylori in Parkinson's Disease Patients May Be Linked to Greater Severity.

Parkinson's disease (PD) is the second most common chronic and progressive neurodegenerative disorder. Its etiology remains elusive and at present only symptomatic treatments exists. Helicobacter pylori chronically colonizes the gastric mucosa of more than half of the global human population. Interestingly, H. pylori positivity has been found to be associated with greater of PD motor severity. In order to investigate the underlying cause of this association, the Sengenics Immunome protein array, which enables simultaneous screening for autoantibodies against 1636 human proteins, was used to screen the serum of 30 H. pylori-seropositive PD patients (case) and 30 age- and gender-matched H. pylori-seronegative PD patients (control) in this study. In total, 13 significant autoantibodies were identified and ranked, with 8 up-regulated and 5 down-regulated in the case group. Among autoantibodies found to be elevated in H. pylori-seropositive PD were included antibodies that recognize Nuclear factor I subtype A (NFIA), Platelet-derived growth factor B (PDGFB) and Eukaryotic translation initiation factor 4A3 (eIFA3). The presence of elevated autoantibodies against proteins essential for normal neurological functions suggest that immunomodulatory properties of H. pylori may explain the association between H. pylori positivity and greater PD motor severity.

Extrahepatic platelet-derived growth factor-ß, delivered by platelets, promotes activation of hepatic stellate cells and biliary fibrosis in mice.

BACKGROUND & AIMS: Platelet-derived growth factor-ß (PDGFB) is a mitogen for hepatic stellate cells (HSCs). We studied the cellular sources of PDGFB and the effects of a high-affinity monoclonal antibody against PDGFB (MOR8457) in mouse models of biliary fibrosis. METHODS: Cellular sources of PDGFB were identified using quantitative reverse-transcription polymerase chain reaction, biochemical, and immunohistologic methods. Mice with advanced biliary fibrosis, MDR2(Abcb4)-null mice, and C57Bl/6 (control) mice were placed on 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-supplemented diets and were given weekly intraperitoneal injections of MOR8457. Platelets were depleted from MDR2-null mice by injection of an antibody against CD41, or inhibited with diets containing low-dose aspirin. Liver tissues were collected and analyzed by quantitative reverse-transcription PCR and histologic and biochemical analyses. RESULTS: Levels of PDGFB protein, but not messenger RNA, were increased in fibrotic livers of MDR2-null mice, compared with control mice. Platelet clusters were detected in the hepatic endothelium, in close proximity to HSCs, and were identified as a source of PDGFB protein in MDR2-null mice. Levels of the PDGFB were increased in serum samples from patients with early stages of liver fibrosis of various etiologies (F1-2, n = 16; P < .05), compared with nonfibrotic liver tissue (F0, n = 12). Depletion of platelets from MDR2-null mice normalized hepatic levels of PDGFB within 48 hours, reducing levels of a marker of HSC activation (α-smooth muscle actin) and expression of genes that promote fibrosis. Diets supplemented with low-dose aspirin reduced circulating serum and hepatic levels of PDGFB and significantly reduced progression of fibrosis in MDR2-null mice over 1 year. MOR8457 produced a dose-dependent decrease in liver fibrosis in MDR2-null mice, reducing collagen deposition by 45% and expression of fibrosis-associated genes by 50%, compared with mice given a control antibody. In vitro, platelets activated freshly isolated HSCs (induction of α-smooth muscle actin and fibrosis-associated genes) via a PDGFB-dependent mechanism. MOR8457 also reduced liver fibrosis in mice placed on DDC-supplemented diets. CONCLUSIONS: Platelets produce PDGFB to activate HSC and promote fibrosis in MDR2-null mice and mice on DDC-supplemented diets. Antiplatelet therapy or selective inhibition of PDGFB might reduce biliary fibrosis in patients with liver disease.

Tumour PDGF-BB expression levels determine dual effects of anti-PDGF drugs on vascular remodelling and metastasis.

Anti-platelet-derived growth factor (PDGF) drugs are routinely used in front-line therapy for the treatment of various cancers, but the molecular mechanism underlying their dose-dependent impact on vascular remodelling remains poorly understood. Here we show that anti-PDGF drugs significantly inhibit tumour growth and metastasis in high PDGF-BB-producing tumours by preventing pericyte loss and vascular permeability, whereas they promote tumour cell dissemination and metastasis in PDGF-BB-low-producing or PDGF-BB-negative tumours by ablating pericytes from tumour vessels. We show that this opposing effect is due to PDGF-ß signalling in pericytes. Persistent exposure of pericytes to PDGF-BB markedly downregulates PDGF-ß and inactivation of the PDGF-ß signalling decreases integrin α1ß1 levels, which impairs pericyte adhesion to extracellular matrix components in blood vessels. Our data suggest that tumour PDGF-BB levels may serve as a biomarker for selection of tumour-bearing hosts for anti-PDGF therapy and unsupervised use of anti-PDGF drugs could potentially promote tumour invasion and metastasis.

Hydroxylamine amplified gold nanoparticle-based aptameric system for the highly selective and sensitive detection of platelet-derived growth factor.

This study developed a sensitive and selective aptamer-based chemiluminescent (CL) method for the determination of platelet-derived growth factor (PDGF)-BB using hydroxylamine enlarged gold nanoparticles (Au NPs). Rabbit anti-human PDGF-BB polyclonal antibody was covalently coupled on the 96-well plate that offers reactive N-oxysuccinimide ester (referred to as NOS group) surface. In the presence of target protein, the biotinylated aptamer was captured on the 96-well plate forming an antibody/PDGF-BB/biotinylated aptamer sandwiched complex, which was followed by the assembly of streptavidin coated Au NPs (streptavidin-gold). Au NPs assembled on the surface of 96-well plate reacted with HAuCl(4) and NH(2)OH, which enabled the catalytic deposition of gold metal onto the Au NPs surfaces. A huge number of Au(3+) ions were released from the hydroxylamine enlarged Au NPs after oxidative gold metal dissolution, which was determined by a simple and sensitive luminol CL reaction. The results showed that the detection limit of the assay is 60 pM of PDGF-BB (corresponding to 6 fmol in a 100 µL volume), which compares favorably with those of other PDGF-BB detection techniques. In addition, this aptameric CL biosensor demonstrated extraordinary specificity. And PDGF-BB has been determined in diluted serum indicating the applicability of this assay.

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

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.

Vaccination with platelet-derived growth factor B kinoids inhibits CCl4-induced hepatic fibrosis in mice.

Platelet-derived growth factor B (PDGF-B) plays an essential role in hepatic fibrosis. Inhibition of the PDGF-B signaling in chronically injured livers might represent a potential therapeutic measure for hepatic fibrosis. In this study, we assessed the effects of vaccination against PDGF-B on CCl4-induced liver fibrosis in BALB/c mice. The PDGF-B kinoid immunogens were prepared by cross-linking two PDGF-B-derived B-cell epitope peptides [PDGF-B¹6-(23-38) and PDGF-B¹6-(72-83)] to ovalbumin and keyhole limpet hemocyanin, respectively. Enzyme-linked immunosorbent assay, Western blotting, and NIH3T3 cell proliferation assay verified that immunization with the PDGF-B kinoids elicited the production of high levels of neutralizing anti-PDGF-B autoantibodies. The vaccination markedly alleviated CCl4-induced hepatic fibrosis, as indicated by the lessened morphological alternations and reduced hydroxyproline contents in the mouse livers. Moreover, immunohistochemical staining for proliferating cell nuclear antigen, α-smooth muscle actin, and desmin demonstrated that neutralization of PDGF-B inhibited both the proliferation and the activation of hepatic stellate cells in the fibrotic mouse livers. Taken together, this study demonstrated that vaccination with PDGF-B kinoids significantly suppressed CCl4-induced hepatic fibrosis in mice. Our results suggest that vaccination against PDGF-B might be developed into an effective, convenient, and safe therapeutic measure for the treatment of hepatic fibrosis.

PAR-2 inhibition reverses experimental pulmonary hypertension.

RATIONALE: A hallmark of the vascular remodeling process underlying pulmonary hypertension (PH) is the aberrant proliferation and migration of pulmonary arterial smooth muscle cells (PASMC). Accumulating evidence suggests that mast cell mediators play a role in the pathogenesis of PH. OBJECTIVE: In the present study we investigated the importance of protease-activated receptor (PAR)-2 and its ligand mast cell tryptase in the development of PH. METHODS AND RESULTS: Our results revealed strong increase in PAR-2 and tryptase expression in the lungs of idiopathic pulmonary arterial hypertension (IPAH) patients, hypoxia-exposed mice, and monocrotaline (MCT)-treated rats. Elevated tryptase levels were also detected in plasma samples from IPAH patients. Hypoxia and platelet-derived growth factor (PDGF)-BB upregulated PAR-2 expression in PASMC. This effect was reversed by HIF (hypoxia inducible factor)-1α depletion, PDGF-BB neutralizing antibody, or the PDGF-BB receptor antagonist Imatinib. Attenuation of PAR-2 expression was also observed in smooth muscle cells of pulmonary vessels of mice exposed to hypoxia and rats challenged with MCT in response to Imatinib treatment. Tryptase induced PASMC proliferation and migration as well as enhanced synthesis of fibronectin and matrix metalloproteinase-2 in a PAR-2- and ERK1/2-dependent manner, suggesting that PAR-2-dependent signaling contributes to vascular remodeling by various mechanisms. Furthermore, PAR-2(-/-) mice were protected against hypoxia-induced PH, and PAR-2 antagonist application reversed established PH in the hypoxia mouse model. CONCLUSIONS: Our study identified a novel role of PAR-2 in vascular remodeling in the lung. Interference with this pathway may offer novel therapeutic options for the treatment of PH.

Restoration and reversible expansion of the osteoblastic hematopoietic stem cell niche after marrow radioablation.

Adequate recovery of hematopoietic stem cell (HSC) niches after cytotoxic conditioning regimens is essential to successful bone marrow transplantation. Yet, very little is known about the mechanisms that drive the restoration of these niches after bone marrow injury. Here we describe a profound disruption of the marrow microenvironment after lethal total body irradiation of mice that leads to the generation of osteoblasts restoring the HSC niche, followed by a transient, reversible expansion of this niche. Within 48 hours after irradiation, surviving host megakaryocytes were observed close to the endosteal surface of trabecular bone rather than in their normal parasinusoidal site concomitant with an increased stromal-derived factor-1 level. A subsequent increase in 2 megakaryocyte-derived growth factors, platelet-derived growth factor-beta and basic fibroblast growth factor, induces a 2-fold expansion of the population of N-cadherin-/osteopontin-positive osteoblasts, relative to the homeostatic osteoblast population, and hence, increases the number of potential niches for HSC engraftment. After donor cell engraftment, this expanded microenvironment reverts to its homeostatic state. Our results demonstrate the rapid recovery of osteoblastic stem cell niches after marrow radioablation, provide critical insights into the associated mechanisms, and suggest novel means to manipulate the bone marrow microenvironment to promote HSC engraftment.

Roles of platelet-derived growth factor-B expression in the ventral horn and motor cortex in the spinal cord-hemisected rhesus monkey.

It is well known that platelet-derived growth factor-B (PDGF-B), a member of the neurotrophic factor family, is involved in normal physiological conditions, pathological changes, and neuroregulation following lesions. But the roles of endogenous PDGF-B in neuroregulation following spinal cord injury are far from being well known, especially in primates. This study explored the role of PDGF-B in the spinal cord and motor cortex in rhesus monkeys subjected to cord hemisection. Evaluation of the hindlimb motor function and the cortical somatosensory evoked potentials (CSEP) demonstrated a significant partial recovery from 30 days post-operation (dpo) to 90 dpo. Immunostaining revealed PDGF-B expression in neurons and scattered macrophages in the spinal cord. The number of PDGF-B immunoreactive neurons in the ventral horn of the spinal cord decreased significantly at the injury site at 14 dpo, followed by a rapid increase that surpassed the numbers in the control group at 30 dpo, and remained at these levels until 90 dpo. The protein levels of PDGF-B and platelet-derived growth factor receptor-beta (PDGFR-beta) as assessed by Western blot, as well as the mRNA levels of PDGF-B as assessed by RT-PCR demonstrated a tendency similar to that seen with immunohistochemistry. PDGF-B antibody administration effectively decreased locomotor function in the hindlimbs, especially on the injured side. No PDGF-B immunoreactive cells were detected in the motor cortex. Taken together, the present findings indicate that intrinsic PDGF-B expressed in the spinal cord may play an essential role in neuroregulation in primates following cord hemisection.

Immunostimulatory effects of low-dose cyclophosphamide are controlled by inducible nitric oxide synthase.

Cyclophosphamide is a widely used chemotherapeutic drug that was recently applied as either an antiangiogenic/antivasculogenic or an immunostimulatory agent in combination with cancer immunotherapies. It has been previously shown that cyclophosphamide augments the efficacy of antitumor immune responses by depleting CD4+ CD25+ T regulatory cells and increasing both T-lymphocyte proliferation and T memory cells. Furthermore, cyclophosphamide was shown to mediate killing of circulating endothelial progenitors. However, the molecular basis for these observations has not yet been elucidated. We show here that the cyclophosphamide-mediated inhibition of inducible nitric oxide synthase is directly linked to its immunostimulatory but not to its antivasculogenic effects. Moreover, combined application of cyclophosphamide with a novel, oral DNA vaccine targeting platelet-derived growth factor B (PDGF-B), overexpressed by proliferating endothelial cells in the tumor vasculature, not only completely inhibited the growth of different tumor types but also led to tumor rejections in mice. These findings provide a new rationale at the molecular level for the combination of chemotherapy and immunotherapy in cancer treatment.

Activation of STAT3/Smad1 is a key signaling pathway for progression to glomerulosclerosis in experimental glomerulonephritis.

Mesangial cell proliferation is a significant event in the development of progressive glomerular injuries. However, the issue of how cell proliferation is involved in the development of glomerulosclerosis is unclear. Recently, we showed that the overexpression of type IV collagen (Col IV), a major component of mesangial extracellular matrix, is transcriptionally regulated by Smad1 in diabetic glomerulosclerosis. In this study, we have demonstrated the effect of the administration of an anti-platelet-derived growth factor (PDGF) beta-receptor antibody (APB5) blocking activation by the PDGF-B chain on rat glomerulonephritis and have examined the signaling pathways that regulate both glomerular cell proliferation and glomerulosclerosis in vivo and in vitro. Experimental mesangial proliferative glomerulonephritis (Thy1 GN) was induced by a single intravenous injection of anti-rat Thy-1.1 monoclonal antibody. In Thy1 GN, mesangial cell proliferation and the expression of Col IV peaked at day 6. Immunohistochemical staining for the expression of Smad1, phospho-Smad1 (pSmad1), and phospho-STAT3 (pSTAT3) revealed that the peak for glomerular Smad1 expression occurred at day 6, consistent with the peak for mesangial proliferation. The expression of pSmad1 was up-regulated at day 1, and the peak for glomerular pSmad1 expression occurred at day 4 of the disease. When treated with APB5, both mesangial proliferation and sclerosis were reduced significantly. The expression of Smad1, pSmad1, and pSTAT3 was also significantly reduced by the administration of APB5. PDGF induced both mesangial cell replication and Col IV synthesis in association with an increased expression of pSTAT3 and pSmad1 on cultured mesangial cells. In addition, APB5 reduced mesangial cell proliferation in association with decreased pSmad1, pSTAT3, and Col IV protein expressions in vitro. The introduction of dominant negative STAT3 significantly decreased the expression of Col IV in cultured mesangial cells. These data suggest that the activation of STAT3 and Smad1 participates in the developing process of glomerulosclerosis in experimental glomerulonephritis.

Adenovirus encoding human platelet-derived growth factor-B delivered in collagen exhibits safety, biodistribution, and immunogenicity profiles favorable for clinical use.

We have developed a therapeutic approach to wound repair involving immobilization of gene transfer vectors within biocompatible matrices (gene-activated matrix, or GAM). The matrix also serves as a scaffold for cellular in-growth and subsequent gene uptake and expression. An adenoviral vector encoding human platelet-derived growth factor-B delivered in collagen (AdPDGF-B/GAM) has demonstrated efficacy in models of wound repair. The safety, biodistribution, and immunogenicity profiles of AdPDGF-B/GAM were examined using a rabbit dermal wound model. Four weekly doses at 1 x 10(10) and 1 x 10(11) viral particles/cm2 of wound surface stimulated dose-related increases in granulation tissue formation and cell proliferation. In situ hybridization and immunostaining demonstrated concordant expression of human PDGF-B mRNA and protein. No treatment-related changes in hematology, serum chemistry, or histopathology were observed. Although AdPDGF-B DNA and PDGF-B mRNA were detected in wounds and axillary lymph nodes of treated animals, no AdPDGF-B was detected in blood or other organs. No immunologic responses against collagen were observed; however, as expected, IgG responses to AdPDGF-B and human PDGF-BB protein were detected. In adenovirus-preimmunized rats, attenuation of the wound healing response was modest (approximately 16%). Collectively, these observations indicate that repeat doses of AdPDGF-B/GAM are well tolerated and lead to robust, localized tissue repair.

Inhibition of platelet-derived growth factor signalling induces autophagy in malignant glioma cells.

Malignant gliomas highly coexpress platelet-derived growth factor (PDGF) and its receptor, suggesting the presence of an autocrine loop. Therefore, disruption of PDGF ligand/receptor complex represents a promising strategy for the treatment of malignant gliomas. However, the mechanisms of the antitumour effect exerted by the inhibition of PDGF-mediated cell growth remain unclear. In the present study, using anti-PDGF neutralising antibody, we investigated the effect of the inhibition of PDGF signalling on malignant glioma U87-MG, D54, and T98G cells with high levels of PDGF-A and -B. As a control, normal fibroblast MRC5 cells expressing low levels of PDGF-A and -B were used. Treatment with anti-PDGF neutralising antibody did not affect the expressions of PDGF-A, PDGF-B, and Akt, but suppressed the level of phosphorylated Akt in tumour cells, indicating the inhibition of PDGF signalling. The cell viability of all malignant glioma cells tested in this study was significantly inhibited in a time-dependent manner following the treatment compared to that of fibroblast cells (P<0.02 to <0.05). The antitumour effect of anti-PDGF antibody was suppressed by the activation of Akt and enhanced by the downregulation of Akt. Interestingly, the inhibition of PDGF signalling induced the development of acidic vesicular organelles and the autophagosome membrane association of the microtubule-associated protein light chain 3, which are characteristic of autophagy, in malignant glioma cells, while apoptotic cell death was not observed. Together these findings imply a new concept of autophagy for PDGF autocrine inhibition in malignant gliomas.