Natural Killer Cell Transcript 4 promotes the development of SjÓ§gren's syndrome via activation of Rap1 on B cells.

Autoimmune disorders are the third most common diseases in the United States, and affect the daily lives of millions of people. In this study, we analyzed patient samples, utilized a transgenic mouse model and human B cells to reveal Natural Killer Cell Transcript 4 (NK4) as a novel regulator that promotes the development of autoimmune disorders. NK4 was significantly elevated in samples from patients with SjÓ§gren's Syndrome (SS). SS patients show elevated NK4 levels. There is a strong and positive correlation between the increased levels of NK4 and the duration of SS. Interestingly, transgenic expression of NK4 in a mouse model led to the development of autoantibodies and lymphocytic infiltration in salivary glands similar to those in SS patients. Those phenotypes were associated with increased B1a cells in the peritoneum, plasma cells in the spleen, and increased IgM, IgA, and IgG2a in serum of the NK4 transgenic mice. The autoimmune phenotypes became more severe in older mice. Moreover, after NK4 transfection, human naïve B cells were activated and memory B cells differentiation into IgG and IgA-plasmablasts, resulting in an increased production of autoantibodies.NK4 regulated the differentiation and activation of B cells through activating Rap1 activity. NK4 also promoted B cell migration in a paracrine fashion through an induction of CXCL13 in endothelial cells. Collectively, these findings identify NK4 as a promoter of the development of autoimmune disorders through its roles on B cells. Therefore, NK4 may be a novel therapeutic target for the treatment of autoimmune diseases.

The Rho/Rac Guanine Nucleotide Exchange Factor Vav1 Regulates Hif-1α and Glut-1 Expression and Glucose Uptake in the Brain.

Vav1 is a Rho/Rac (Ras-related C3 botulinum toxin substrate) guanine nucleotide exchange factor expressed in hematopoietic and endothelial cells that are involved in a wide range of cellular functions. It is also stabilized under hypoxic conditions when it regulates the accumulation of the transcription factor HIF (Hypoxia Inducible Factor)-1α, which activates the transcription of target genes to orchestrate a cellular response to low oxygen. One of the genes induced by HIF-1α is GLUT (Glucose Transporter)-1, which is the major glucose transporter expressed in vessels that supply energy to the brain. Here, we identify a role for Vav1 in providing glucose to the brain. We found that Vav1 deficiency downregulates HIF-1α and GLUT-1 levels in endothelial cells, including blood-brain barrier cells. This downregulation of GLUT-1, in turn, reduced glucose uptake to endothelial cells both in vitro and in vivo, and reduced glucose levels in the brain. Furthermore, endothelial cell-specific Vav1 knock-out in mice, which caused glucose uptake deficiency, also led to a learning delay in fear conditioning experiments. Our results suggest that Vav1 promotes learning by activating HIF-1α and GLUT-1 and thereby distributing glucose to the brain. We further demonstrate the importance of glucose transport by endothelial cells in brain functioning and reveal a potential new axis for targeting GLUT-1 deficiency syndromes and other related brain diseases.

Attenuation of Myeloid-Specific TGFß Signaling Induces Inflammatory Cerebrovascular Disease and Stroke.

RATIONALE: Cryptogenic strokes, those of unknown cause, have been estimated as high as 30% to 40% of strokes. Inflammation has been suggested as a critical etiologic factor. However, there is lack of experimental evidence. OBJECTIVE: In this study, we investigated inflammation-associated stroke using a mouse model that developed spontaneous stroke because of myeloid deficiency of TGF-ß (transforming growth factor-ß) signaling. METHODS AND RESULTS: We report that mice with deletion of Tgfbr2 in myeloid cells (Tgfbr2Myeko) developed cerebrovascular inflammation in the absence of significant pathology in other tissues, culminating in stroke and severe neurological deficits with 100% penetrance. The stroke phenotype can be transferred to syngeneic wild-type mice via Tgfbr2Myeko bone marrow transplant and can be rescued in Tgfbr2Myeko mice with wild-type bone marrow. The underlying mechanisms involved an increased type 1 inflammation and cerebral endotheliopathy, characterized by elevated NF-κB (nuclear factor-κB) activation and TNF (tumor necrosis factor) production by myeloid cells. A high-fat diet accelerated stroke incidence. Anti-TNF treatment, as well as metformin and methotrexate, which are associated with decreased stroke risk in population studies, delayed stroke occurrence. CONCLUSIONS: Our studies show that TGF-ß signaling in myeloid cells is required for maintenance of vascular health and provide insight into inflammation-mediated cerebrovascular disease and stroke.

Mechanisms that drive inflammatory tumor microenvironment, tumor heterogeneity, and metastatic progression.

Treatment of cancer metastasis has been largely ineffective. It is paramount to understand the mechanisms underlying the metastatic process, of which the tumor microenvironment is an indispensable participant. What are the critical cellular and molecular players at the primary tumor site where metastatic cascade initiates? How is tumor-associated inflammation regulated? How do altered vasculatures contribute to metastasis? What is the dynamic nature or heterogeneity of primary tumors and what are the challenges to catch a moving target? This review summarizes recent progress, mechanistic understanding, and options for metastasis-targeted therapy.

Differential regulation of blood flow-induced neovascularization and mural cell recruitment by vascular endothelial growth factor and angiopoietin signalling.

KEY POINTS: Combining nitric oxide (NO)-mediated increased blood flow with angiopoietin-1-Tie2 receptor signalling induces arteriolargenesis - the formation of arterioles from capillaries - in a model of physiological angiogenesis. This NO-Tie-mediated arteriolargenesis requires endogenous vascular endothelial growth factor (VEGF) signalling. Inhibition of VEGF signalling increases pericyte coverage in microvessels. Together these findings indicate that generation of functional neovasculature requires close titration of NO-Tie2 signalling and localized VEGF induction, suggesting that the use of exogenous VEGF expression as a therapeutic for neovascularization may not be successful. ABSTRACT: Signalling through vascular endothelial growth factor (VEGF) receptors and the tyrosine kinase with IgG and EGF domains-2 (Tie2) receptor by angiopoietins is required in combination with blood flow for the formation of a functional vascular network. We tested the hypothesis that VEGF and angiopoietin-1 (Ang1) contribute differentially to neovascularization induced by nitric oxide (NO)-mediated vasodilatation, by comparing the phenotype of new microvessels in the mesentery during induction of vascular remodelling by over-expression of endothelial nitric oxide synthase in the fat pad of the adult rat mesentery during inhibition of angiopoietin signalling with soluble Tie2 (sTie2) and VEGF signalling with soluble Fms-like tyrosine kinase receptor-1 (sFlt1). We found that NO-mediated angiogenesis was blocked by inhibition of VEGF with sFlt1 (from 881 ± 98% increase in functional vessel area to 279 ± 72%) and by inhibition of angiopoietin with sTie2 (to 337 ± 67%). Exogenous angiopoietin-1 was required to induce arteriolargenesis (8.6 ± 1.3% of vessels with recruitment of vascular smooth muscle cells; VSMCs) in the presence of enhanced flow. sTie2 and sFlt1 both inhibited VSMC recruitment (both 0%), and VEGF inhibition increased pericyte recruitment to newly formed vessels (from 27 ± 2 to 54 ± 3% pericyte ensheathment). We demonstrate that a fine balance of VEGF and angiopoietin signalling is required for the formation of a functional vascular network. Endogenous VEGF signalling prevents excess neovessel pericyte coverage, and is required for VSMC recruitment during increased nitric oxide-mediated vasodilatation and angiopoietin signalling (NO-Tie-mediated arteriogenesis). Therapeutic vascular remodelling paradigms may therefore require treatments that modulate blood flow to utilize endogenous VEGF, in combination with exogenous Ang1, for effective neovascularization.

Vav1 Regulates Mesenchymal Stem Cell Differentiation Decision Between Adipocyte and Chondrocyte via Sirt1.

Mesenchymal stem cells (MSCs) are multipotent stromal cells residing in the bone marrow. MSCs have the potential to differentiate to adipocytes, chondrocytes, and other types of cells. In this study, we investigated the molecular mechanism that controls MSC cell fate decisions for differentiation. We found that Vav1, a guanine nucleotide exchange factor for Rho GTPase, was highly expressed in MSCs. Interestingly, loss of Vav1 in MSCs led to spontaneous adipogenic but impaired chondrogenic differentiation, and accordingly Vav1 null mice displayed an increase in fat content and a decrease in cartilage. Conversely, ectopic expression of Vav1 in MSCs reversed this phenotype, and led to enhanced MSC differentiation into chondrocyte but retarded adipogenesis. Mechanistically, loss of Vav1 reduced the level of Sirt1, which was responsible for an increase of acetylated PPARγ. As acetylation activates PPARγ, it increased C/EBPα expression and promoted adipogenesis. On the other hand, loss of Vav1 resulted in an increase of acetylated Sox9, a target of Sirt1. As acetylation represses Sox9 activity, it led to a dramatic reduction of collagen 2α1, a key regulator in chondrocyte differentiation. Finally, we found that Vav1 regulates Sirt1 in MSCs through Creb. Together this study reveals a novel function of Vav1 in regulating MSC cell fate decisions for differentiation through Sirt1. Sirt1 deacetylates PPARγ and Sox9, two key mediators that control adipocyte and chondrocyte differentiation. The acetylation status of PPARγ and Sox9 has opposite effects on its activity, thereby controlling cell fate decision. Stem Cells 2016;34:1934-1946.

Macrophage IKKα Deficiency Suppresses Akt Phosphorylation, Reduces Cell Survival, and Decreases Early Atherosclerosis.

OBJECTIVE: The IκB kinase (IKK) is an enzyme complex that initiates the nuclear factor κB transcription factor cascade, which is important in regulating multiple cellular responses. IKKα is directly associated with 2 major prosurvival pathways, PI3K/Akt and nuclear factor κB, but its role in cell survival is not clear. Macrophages play critical roles in the pathogenesis of atherosclerosis, yet the impact of IKKα signaling on macrophage survival and atherogenesis remains unclear. APPROACH AND RESULTS: Here, we demonstrate that genetic IKKα deficiency, as well as pharmacological inhibition of IKK, in mouse macrophages significantly reduces Akt S(473) phosphorylation, which is accompanied by suppression of mTOR complex 2 signaling. Moreover, IKKα null macrophages treated with lipotoxic palmitic acid exhibited early exhaustion of Akt signaling compared with wild-type cells. This was accompanied by a dramatic decrease in the resistance of IKKα(-/-) monocytes and macrophages to different proapoptotic stimuli compared with wild-type cells. In vivo, IKKα deficiency increased macrophage apoptosis in atherosclerotic lesions and decreased early atherosclerosis in both female and male low-density lipoprotein receptor (LDLR)(-/-) mice reconstituted with IKKα(-/-) hematopoietic cells and fed with the Western diet for 8 weeks compared with control LDLR(-/-) mice transplanted with wild-type cells. CONCLUSIONS: Hematopoietic IKKα deficiency in mouse suppresses Akt signaling, compromising monocyte/macrophage survival and this decreases early atherosclerosis.

Abrogation of TGF beta signaling in mammary carcinomas recruits Gr-1+CD11b+ myeloid cells that promote metastasis.

Aberrant TGFbeta signaling is common in human cancers and contributes to tumor metastasis. Here, we demonstrate that Gr-1+CD11b+ myeloid cells are recruited into mammary carcinomas with type II TGF beta receptor gene (Tgfbr2) deletion and directly promote tumor metastasis. Gr-1+CD11b+ cells infiltrate into the invasive front of tumor tissues and facilitate tumor cell invasion and metastasis through a process involving metalloproteinase activity. This infiltration of Gr-1+CD11b+ cells also results in increased abundance of TGF beta 1 in tumors with Tgfbr2 deletion. The recruitment of Gr-1+CD11b+ cells into tumors with Tgfbr2 deletion involves two chemokine receptor axes, the SDF-1/CXCR4 and CXCL5/CXCR2 axes. Together, these data indicate that Gr-1+CD11b+ cells contribute to TGFbeta-mediated metastasis through enhancing tumor cell invasion and metastasis.

Structures of a platelet-derived growth factor/propeptide complex and a platelet-derived growth factor/receptor complex.

Platelet-derived growth factors (PDGFs) and their receptors (PDGFRs) are prototypic growth factors and receptor tyrosine kinases which have critical functions in development. We show that PDGFs share a conserved region in their prodomain sequences which can remain noncovalently associated with the mature cystine-knot growth factor domain after processing. The structure of the PDGF-A/propeptide complex reveals this conserved, hydrophobic association mode. We also present the structure of the complex between PDGF-B and the first three Ig domains of PDGFRbeta, showing that two PDGF-B protomers clamp PDGFRbeta at their dimerization seam. The PDGF-B:PDGFRbeta interface is predominantly hydrophobic, and PDGFRs and the PDGF propeptides occupy overlapping positions on mature PDGFs, rationalizing the need of propeptides by PDGFs to cover functionally important hydrophobic surfaces during secretion. A large-scale structural organization and rearrangement is observed for PDGF-B upon receptor binding, in which the PDGF-B L1 loop, disordered in the structure of the free form, adopts a highly specific conformation to form hydrophobic interactions with the third Ig domain of PDGFRbeta. Calorimetric data also shows that the membrane-proximal homotypic PDGFRalpha interaction, albeit required for activation, contributes negatively to ligand binding. The structural and biochemical data together offer insights into PDGF-PDGFR signaling, as well as strategies for PDGF-antagonism.

Simultaneous on-chip DC dielectrophoretic cell separation and quantitative separation performance characterization.

Through integration of a MOSFET-based microfluidic Coulter counter with a dc-dielectrophoretic cell sorter, we demonstrate simultaneous on-chip cell separation and sizing with three different samples including 1) binary mixtures of polystyrene beads, 2) yeast cells of continuous size distribution, and 3) mixtures of 4T1 tumor cells and murine bone marrow cells. For cells with continuous size distribution, it is found that the receiver operator characteristic analysis is an ideal method to characterize the separation performance. The characterization results indicate that dc-DEP separation performance degrades as the sorting throughput (cell sorting rate) increases, which provides insights into the design and operation of size-based microfluidic cell separation.

Identification of a myeloid-derived suppressor cell cystatin-like protein that inhibits metastasis.

Myeloid-derived suppressor cells (MDSCs) are significantly increased in cancer patients and tumor bearing-animals. MDSCs infiltrate into tumors and promote tumor invasion and metastasis. To identify the mediator responsible for the prometastatic property of MDSCs, we used proteomics. We found neutrophilic granule protein (NGP) was decreased >2-fold in MDSCs from metastatic 4T1 tumor-bearing mice compared to nonmetastatic 67NR controls. NGP mRNA levels were decreased in bone marrow and in tumor-infiltrating MDSCs by 45 and 66%, respectively, in 4T1 tumor-bearing mice compared to 67NR controls. Interestingly, 4T1-conditioned medium reduced myeloid cell NGP expression by ∼ 40%, suggesting that a secreted factor mediates gene reduction. Sequence analysis shows a putative cystatin domain in NGP, and biochemical analysis confirms NGP a novel cathepsin inhibitor. It inhibited cathepsin B activity by nearly 40% in vitro. NGP expression in 4T1 tumor cells suppressed cell invasion, delayed primary tumor growth, and greatly reduced lung metastasis in vivo. A 2.8-fold reduction of cathepsin activity was found in tumors expressing NGP compared to controls. NGP significantly reduced tumor angiogenesis to 12.6 from 19.6 and lymphangiogenesis to 4.6 from 9.1 vessels/field. Necrosis was detectable only in NGP-expressing tumors, and the number of apoptotic cells increased to 22.4 from 8.3 in controls. Taken together, this study identifies a negative regulator of tumor metastasis in MDSCs, NGP, which is down-regulated in metastatic conditions. The finding suggests that malignant tumors promote invasion/metastasis not only through up-regulation of proteases but also down-regulation of protease inhibitors.

Negative regulation of myeloid-derived suppressor cells in cancer.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells with suppressive function on immune response. In this review, we discuss recent studies about mechanisms of expansion and suppressive function of MDSCs during inflammation, infection and autoimmune diseases, as well as pro-angiogenic and pro-metastatic functions of these cells in tumor development. Further, we focus on novel studies of MDSCs and therapeutic approaches to eliminate these cells in cancer.

Expansion of myeloid immune suppressor Gr+CD11b+ cells in tumor-bearing host directly promotes tumor angiogenesis.

We demonstrate a novel tumor-promoting role of myeloid immune suppressor Gr+CD11b+ cells, which are evident in cancer patients and tumor-bearing animals. These cells constitute approximately 5% of total cells in tumors. Tumors coinjected with Gr+CD11b+ cells exhibited increased vascular density, vascular maturation, and decreased necrosis. These immune cells produce high levels of MMP9. Deletion of MMP9 in these cells completely abolishes their tumor-promoting ability. Gr+CD11b+ cells were also found to directly incorporate into tumor endothelium. Consistent with this observation, Gr+CD11b+ cells acquire endothelial cell (EC) properties in tumor microenvironment and proangiogenic culture conditions. Our data provide evidence that Gr+CD11b+ cells of immune origin induced by tumors directly contribute to tumor growth and vascularization by producing MMP9 and differentiating into ECs.

A versatile valve-enabled microfluidic cell co-culture platform and demonstration of its applications to neurobiology and cancer biology.

A versatile microfluidic platform allowing co-culture of multiple cell populations in close proximity with separate control of their microenvironments would be extremely valuable for many biological applications. Here, we report a simple and compact microfluidic platform that has these desirable features and allows for real-time, live-cell imaging of cell-cell interactions. Using a pneumatically/hydraulically controlled poly(dimethylsiloxane) (PDMS) valve barrier, distinct cell types can be cultured in side-by-side microfluidic chambers with their optimum culture media and treated separately without affecting the other cell population. The platform is capable of both two-dimensional and three-dimensional cell co-culture and through variations of the valve barrier design, the platform allows for cell-cell interactions through either direct cell contact or soluble factors alone. The platform has been used to perform dynamic imaging of synapse formation in hippocampal neurons by separate transfection of two groups of neurons with fluorescent pre- and post-synaptic protein markers. In addition, cross-migration of 4T1 tumor cells and endothelial cells has been studied under normoxic and hypoxic conditions, which revealed different migration patterns, suggesting the importance of the microenvironments in cell-cell interactions and biological activities.

Vav1 is Essential for HIF-1α Activation via a Lysosomal VEGFR1-Mediated Degradation Mechanism in Endothelial Cells.

The vascular response to hypoxia and ischemia is essential for maintaining homeostasis during stressful conditions and is particularly critical for vital organs such as the heart. Hypoxia-inducible factor-1 (HIF-1) is a central regulator of the response to hypoxia by activating transcription of numerous target genes, including vascular endothelial growth factor (VEGF). Here we identify the guanine nucleotide exchange factor (GEF) Vav1, a regulator of the small Rho-GTPase and cell signaling in endothelial cells, as a key vascular regulator of hypoxia. We show that Vav1 is present in the vascular endothelium and is essential for HIF-1 activation under hypoxia. So, we hypothesized that Vav1 could be a key regulator of HIF-1 signaling. In our findings, Vav1 regulates HIF-1α stabilization through the p38/Siah2/PHD3 pathway. In normoxia, Vav1 binds to vascular endothelial growth factor receptor 1 (VEGFR1), which directs Vav1 to lysosomes for degradation. In contrast, hypoxia upregulates Vav1 protein levels by inhibiting lysosomal degradation, which is analogous to HIF-1α regulation by hypoxia: both proteins are constitutively produced and degraded in normoxia allowing for a rapid response when stress occurs. Consequently, hypoxia rapidly stabilizes Vav1, which is required for HIF-1α accumulation. This shows that Vav1 is the key mediator controlling the stabilization of HIF1α in hypoxic conditions. With this finding, we report a novel pathway to stabilize HIF-1, which shows a possible reason why clinical trials targeting HIF-1 has not been effective. Targeting Vav1 can be the new approach to overcome hypoxic tumors.

Molecular characterization of IL-32 in human endothelial cells.

IL-32 is a newly discovered protein found in human and certain primates, but absent in rodent. Various reports suggest its role as a proinflammatory mediator. Since vascular endothelium is critical in inflammation, we investigate IL-32 in endothelial cells. We found that the gene is expressed in human endothelial cells and Akt strongly induces its expression. Sequence analysis indicates IL-32 beta as the major isoform in endothelial cells. Surprisingly, we did not detect any secretion of IL-32 beta in human endothelial cells; instead we observed co-localization of IL-32 beta with endoplasmic reticulum, suggesting IL-32 beta is an intracellular protein in these cells. Promoter analysis identified a minimum required region for IL-32 transcription at -0.1 to +0.5 kb around the initially identified transcription start site. We also defined a transcriptional suppressor-binding site at -2.0 to -1.5 kb. Importantly, RNA ligase mediated rapid amplification of cDNA ends in endothelial cells determined the transcription start site at the 328 bp downstream from the original identified site. Finally, we found a positive correlation of IL-32 levels with human breast cancer and glioblastoma multiforme (GBM). These findings improve our understanding of IL-32 in vascular endothelium. IL-32 expression might be valuable as a biomarker for cancer.

Angiogenesis links chronic inflammation with cancer.

Angiogenesis, the formation of new blood vessels from existing vessels, is tightly linked to chronic inflammation and cancer. Angiogenesis is one of the molecular events bridging the gap between inflammation and cancer. One of the events linking inflammation and cancer is an increase in cellular adhesion molecules that are expressed on the luminal surface of endothelium upon inflammation. Cellular adhesion molecules are involved in leukocyte recruitment and subsequently lead to extravasation of leukocytes to the injury site. These adhesion molecules are known to be shared by some cancer cells and have the ability to contribute to metastasis. Thus, an elevation of these molecules in chronic inflammation may be a risk factor for metastasis. In this chapter, we discuss the method used to determine the adhesion molecules expressed on endothelium, and leukocyte adhesion to endothelium.

Interleukin-33 and ST2 Signaling in Tumor Microenvironment.

Interleukin-33 (IL-33) is one of the members of the IL-1 family of cytokines and a ligand of ST2 and IL-1 receptor accessory protein (IL-1RAcP) that is known to affect Th2 inflammatory response with partial effects on Th1 responses. This cytokine is released by epithelial and smooth muscle cells of the airway system during their injury by several environmental stimuli, such as allergens, viruses, helminths, and pollutants. IL-33 is an alarmin that acts as an endogenous danger signal, and it has been known to affect various types of cells, such as mast cells, basophils, eosinophils, T cells, and specific subsets of innate lymphoid cells (ILCs). In recent findings, this cytokine is believed to have a critical role in several types of cancers, such as lung cancer, liver cancer, and head and neck squamous cell cancer. The expression of IL-33/ST2 in cancer tissues shows a close association with tumor growth and tumor progression in several types of cancer, suggesting the IL-33/ST2 pathway as a potential target for therapy.

Oxygen Tension Regulates Lysosomal Activation and Receptor Tyrosine Kinase Degradation.

Oxygen sensing is crucial for adaptation to variable habitats and physiological conditions. Low oxygen tension, or hypoxia, is a common feature of solid tumors, and hypoxic tumors are often more aggressive and resistant to therapy. Here we show that, in cultured mammalian cells, hypoxia suppressed lysosomal acidification/activation and receptor tyrosine kinase (RTK) degradation. Hypoxia down-regulated mTORc1, reducing its ability to activate transcription factor EB (TFEB), a master regulator of V-ATPase, the lysosomal proton pump. Hypoxia prevented epidermal growth factor receptor (EGFR) degradation in tumor tissues, whereas activation of lysosomes enhanced tumor cell response to anti-EGFR treatment. Our results link oxygen tension and lysosomal activity, provide a molecular explanation of the malignant phenotype associated with hypoxic tumors, and suggest activation of lysosomes may provide therapeutic benefit in RTK-targeted cancer therapy.

Neutrophilic Granule Protein Is a Novel Murine LPS Antagonist.

Neutrophilic granule protein (NGP) was previously reported as a granular protein of neutrophils in mouse, but the function has not been known clearly. We found the presence of the possible signal peptide in NGP and validated this protein is circulating in the bloodstream. In our findings, NGP is being modified post-translationally in Golgi apparatus and endoplasmic reticulum, which is a universal character of secretory molecules with a signal peptide. The secreted NGP protein could be detected both in vitro and in vivo. NGP has sequence similarity with an antimicrobial protein cathelicidin, and we observed the aspect of inflammation of NGP. Interestingly, NGP interacts with the complex of LPS and LPS binding protein (LBP). This interaction blocks the binding of the complex of LPS and LBP to TLR4 and the downstream inflammatory signals. Furthermore, the inhibitory function of NGP against the inflammatory effect of LPS could be observed in both in vitro and in vivo. With these findings, we report NGP is a novel secretory protein to mask LPS and inhibit its function.