Enhanced delivery of antibodies across the blood-brain barrier via TEMs with inherent receptor-mediated phagocytosis.

BACKGROUND: The near impermeability of the blood-brain barrier (BBB) and the unique neuroimmune environment of the CNS prevents the effective use of antibodies in neurological diseases. Delivery of biotherapeutics to the brain can be enabled through receptor-mediated transcytosis via proteins such as the transferrin receptor, although limitations such as the ability to use Fc-mediated effector function to clear pathogenic targets can introduce safety liabilities. Hence, novel delivery approaches with alternative clearance mechanisms are warranted. METHODS: Binders that optimized transport across the BBB, known as transcytosis-enabling modules (TEMs), were identified using a combination of antibody discovery techniques and pharmacokinetic analyses. Functional activity of TEMs were subsequently evaluated by imaging for the ability of myeloid cells to phagocytose target proteins and cells. FINDINGS: We demonstrated significantly enhanced brain exposure of therapeutic antibodies using optimal transferrin receptor or CD98 TEMs. We found that these modules also mediated efficient clearance of tau aggregates and HER2+ tumor cells via a non-classical phagocytosis mechanism through direct engagement of myeloid cells. This mode of clearance potentially avoids the known drawbacks of FcγR-mediated antibody mechanisms in the brain such as the neurotoxic release of proinflammatory cytokines and immune cell exhaustion. CONCLUSIONS: Our study reports a new brain delivery platform that harnesses receptor-mediated transcytosis to maximize brain uptake and uses a non-classical phagocytosis mechanism to efficiently clear pathologic proteins and cells. We believe these findings will transform therapeutic approaches to treat CNS diseases. FUNDING: This research was funded by Janssen, Pharmaceutical Companies of Johnson & Johnson.

NG2 Proteoglycan-Dependent Contributions of Pericytes and Macrophages to Brain Tumor Vascularization and Progression.

The NG2 proteoglycan promotes tumor growth as a component of both tumor and stromal cells. Using intracranial, NG2-negative B16F10 melanomas, we have investigated the importance of PC and Mac NG2 in brain tumor progression. Reduced melanoma growth in Mac-NG2ko and PC-NG2ko mice demonstrates the importance of NG2 in both stromal compartments. In each genotype, the loss of PC-endothelial cell interaction diminishes the formation of endothelial junctions and assembly of the basal lamina. Tumor vessels in Mac-NG2ko mice have smaller diameters, reduced patency, and increased leakiness compared to PC-NG2ko mice, thus decreasing tumor blood supply and increasing hypoxia. While the reduced PC interaction with endothelial cells in PC-NG2ko mice results from the loss of PC activation of ß1 integrin signaling in endothelial cells, reduced PC-endothelial cell interaction in Mac-NG2ko mice results from 90% reduced Mac recruitment. The absence of Mac-derived signals in Mac-NG2ko mice causes the loss of PC association with endothelial cells. Reduced Mac recruitment may be due to diminished activation of integrins in the absence of NG2, causing decreased Mac interaction with endothelial adhesion molecules that are needed for extravasation. These results reflect the complex interplay that occurs between Mac, PC, and endothelial cells during tumor vascularization.

Role of NG2 proteoglycan in macrophage recruitment to brain tumors and sites of CNS demyelination.

Macrophage infiltration is a factor in most if not all inflammatory pathologies. Understanding molecular interactions that underlie this process is therefore important for our ability to modulate macrophage behavior for therapeutic purposes. Our studies show that cell surface expression of the nerveglial antigen 2 (NG2) proteoglycan is important for the ability of macrophages to colonize both brain tumors and sites of central nervous system (CNS) demyelination. Myeloid-specific ablation of NG2 using LysM-Cre deleter mice results in large decreases in macrophage abundance in both an intracranial melanoma model and a lysolecithin model of spinal cord demyelination. In the melanoma model, decreased macrophage recruitment in the NG2 null mice leads to diminished tumor growth. In line with observations in the literature, this phenomenon is based in part on deficits in tumor vascularization that result from loss of pericyte interaction with endothelial cells in the absence of a macrophage-derived factor(s). In the demyelination model, decreased macrophage infiltration in the NG2 null mice is associated with an initial reduction in lesion size, but nevertheless also with deficits in repair of the lesion. Diminished myelin repair is due not only to reduced clearance of myelin debris, but also to decreased proliferation/recruitment of oligodendrocyte progenitor cells in the absence of a macrophage-derived factor(s). Thus, in both models macrophages have secondary effects on other cell types that are important for progression of the specific pathology. Efforts are underway to identify mechanisms by which NG2 influences macrophage recruitment and by which macrophages signal to other cell types involved in the pathologies.

NG2 proteoglycan-dependent recruitment of tumor macrophages promotes pericyte-endothelial cell interactions required for brain tumor vascularization.

Early stage growth of intracranial B16F10 tumors is reduced by 87% in myeloid-specific NG2 null (Mac-NG2ko) mice and by 77% in pericyte-specific NG2 null (PC-NG2ko) mice, demonstrating the importance of the NG2 proteoglycan in each of these stromal compartments. In both genotypes, loss of pericyte-endothelial cell interaction results in numerous structural defects in tumor blood vessels, including decreased formation of endothelial cell junctions and decreased assembly of the vascular basal lamina. All vascular deficits are larger in Mac-NG2ko mice than in PC-NG2ko mice, correlating with the greater decrease in pericyte-endothelial cell interaction in Mac-NG2ko animals. Accordingly, tumor vessels in Mac-NG2ko mice have a smaller diameter, lower degree of patency, and higher degree of leakiness than tumor vessels in PC-NG2ko mice, leading to less efficient tumor blood flow and to increased intratumoral hypoxia. While reduced pericyte interaction with endothelial cells in PC-NG2ko mice is caused by loss of NG2-dependent pericyte activation of ß1 integrin signaling in endothelial cells, reduced pericyte-endothelial cell interaction in Mac-NG2ko mice is due to a 90% reduction in NG2-dependent macrophage recruitment to tumors. The absence of a macrophage-derived signal(s) in Mac-NG2ko mice results in the loss of pericyte ability to associate with endothelial cells, possibly due to reduced expression of N-cadherin by both pericytes and endothelial cells.

Genetic disruption of the sh3pxd2a gene reveals an essential role in mouse development and the existence of a novel isoform of tks5.

Tks5 is a scaffold protein and Src substrate involved in cell migration and matrix degradation through its essential role in invadosome formation and function. We have previously described that Tks5 is fundamental for zebrafish neural crest cell migration in vivo. In the present study, we sought to investigate the function of Tks5 in mammalian development by analyzing mice mutant for sh3pxd2a, the gene encoding Tks5. Homozygous disruption of the sh3pxd2a gene by gene-trapping in mouse resulted in neonatal death and the presence of a complete cleft of the secondary palate. Interestingly, embryonic fibroblasts from homozygous gene-trap sh3pxd2a mice lacked only the highest molecular weight band of the characteristic Tks5 triplet observed in protein extracts, leaving the lower molecular weight bands unaffected. This finding, together with the existence of two human Expressed Sequence Tags lacking the first 5 exons of SH3PXD2A, made us hypothesize about the presence of a second alternative transcription start site located in intron V. We performed 5'RACE on mouse fibroblasts and isolated a new transcript of the sh3pxd2a gene encoding a novel Tks5 isoform, that we named Tks5ß. This novel isoform diverges from the long form of Tks5 in that it lacks the PX-domain, which confers affinity for phosphatidylinositol-3,4-bisphosphate. Instead, Tks5ß has a short unique amino terminal sequence encoded by the newly discovered exon 6ß; this exon includes a start codon located 29 bp from the 5'-end of exon 6. Tks5ß mRNA is expressed in MEFs and all mouse adult tissues analyzed. Tks5ß is a substrate for the Src tyrosine kinase and its expression is regulated through the proteasome degradation pathway. Together, these findings indicate the essentiality of the larger Tks5 isoform for correct mammalian development and the transcriptional complexity of the sh3pxd2a gene.

Podosomal proteins as causes of human syndromes: a role in craniofacial development?

Podosomes and invadopodia are actin-rich protrusions of the plasma membrane important for matrix degradation and cell migration. Most of the information in this field has been obtained in cancer cells, where the presence of invadopodia has been related to increased invasiveness and metastatic potential. The importance of the related podosome structure in other pathological or physiological processes that require cell invasion is relatively unexplored. Recent evidence indicates that essential components of podosomes are responsible for several human syndromes, some of which are characterized by serious developmental defects involving the craniofacial area, skeleton and heart, and very poor prognosis. Here we will review them and discuss the possible role of podosomes as a player in correct embryo development.

Disruption of the podosome adaptor protein TKS4 (SH3PXD2B) causes the skeletal dysplasia, eye, and cardiac abnormalities of Frank-Ter Haar Syndrome.

Frank-Ter Haar syndrome (FTHS), also known as Ter Haar syndrome, is an autosomal-recessive disorder characterized by skeletal, cardiovascular, and eye abnormalities, such as increased intraocular pressure, prominent eyes, and hypertelorism. We have conducted homozygosity mapping on patients representing 12 FTHS families. A locus on chromosome 5q35.1 was identified for which patients from nine families shared homozygosity. For one family, a homozygous deletion mapped exactly to the smallest region of overlapping homozygosity, which contains a single gene, SH3PXD2B. This gene encodes the TKS4 protein, a phox homology (PX) and Src homology 3 (SH3) domain-containing adaptor protein and Src substrate. This protein was recently shown to be involved in the formation of actin-rich membrane protrusions called podosomes or invadopodia, which coordinate pericellular proteolysis with cell migration. Mice lacking Tks4 also showed pronounced skeletal, eye, and cardiac abnormalities and phenocopied the majority of the defects associated with FTHS. These findings establish a role for TKS4 in FTHS and embryonic development. Mutation analysis revealed five different homozygous mutations in SH3PXD2B in seven FTHS families. No SH3PXD2B mutations were detected in six other FTHS families, demonstrating the genetic heterogeneity of this condition. Interestingly however, dermal fibroblasts from one of the individuals without an SH3PXD2B mutation nevertheless expressed lower levels of the TKS4 protein, suggesting a common mechanism underlying disease causation.

The novel adaptor protein Tks4 (SH3PXD2B) is required for functional podosome formation.

Metastatic cancer cells have the ability to both degrade and migrate through the extracellular matrix (ECM). Invasiveness can be correlated with the presence of dynamic actin-rich membrane structures called podosomes or invadopodia. We showed previously that the adaptor protein tyrosine kinase substrate with five Src homology 3 domains (Tks5)/Fish is required for podosome/invadopodia formation, degradation of ECM, and cancer cell invasion in vivo and in vitro. Here, we describe Tks4, a novel protein that is closely related to Tks5. This protein contains an amino-terminal Phox homology domain, four SH3 domains, and several proline-rich motifs. In Src-transformed fibroblasts, Tks4 is tyrosine phosphorylated and predominantly localized to rosettes of podosomes. We used both short hairpin RNA knockdown and mouse embryo fibroblasts lacking Tks4 to investigate its role in podosome formation. We found that lack of Tks4 resulted in incomplete podosome formation and inhibited ECM degradation. Both phenotypes were rescued by reintroduction of Tks4, whereas only podosome formation, but not ECM degradation, was rescued by overexpression of Tks5. The tyrosine phosphorylation sites of Tks4 were required for efficient rescue. Furthermore, in the absence of Tks4, membrane type-1 matrix metalloproteinase (MT1-MMP) was not recruited to the incomplete podosomes. These findings suggest that Tks4 and Tks5 have overlapping, but not identical, functions, and implicate Tks4 in MT1-MMP recruitment and ECM degradation.

Cutting edge: Essential role of hypoxia inducible factor-1alpha in development of lipopolysaccharide-induced sepsis.

Sepsis, the leading cause of death in intensive care units, reflects a detrimental host response to infection in which bacteria or LPS act as potent activators of immune cells, including monocytes and macrophages. In this report, we show that LPS raises the level of the transcriptional regulator hypoxia-inducible factor-1alpha (HIF-1alpha) in macrophages, increasing HIF-1alpha and decreasing prolyl hydroxylase mRNA production in a TLR4-dependent fashion. Using murine conditional gene targeting of HIF-1alpha in the myeloid lineage, we demonstrate that HIF-1alpha is a critical determinant of the sepsis phenotype. HIF-1alpha promotes the production of inflammatory cytokines, including TNF-alpha, IL-1, IL-4, IL-6, and IL-12, that reach harmful levels in the host during early sepsis. HIF-1alpha deletion in macrophages is protective against LPS-induced mortality and blocks the development of clinical markers including hypotension and hypothermia. Inhibition of HIF-1alpha activity may thus represent a novel therapeutic target for LPS-induced sepsis.

Increased expression of the renin-angiotensin system and mast cell density but not of angiotensin-converting enzyme II in late stages of human heart failure.

BACKGROUND: The activation of the renin-angiotensin system (RAS) contributes to the progression of left ventricular dysfunction. A novel human homologue of the angiotensin-converting enzyme (ACE), named ACE2, has been described but its role in human heart failure (HF) has not been elucidated. Besides, there is controversy as to whether the major angiotensin II-forming-activity in heart is ACE or chymase released from mast cells. Furthermore, long-term blockade of nitric oxide (NO) synthesis has been shown to increase ACE activity. To assess the locally activated vasoactive mediators that may contribute to the ventricular deterioration process, we sought to simultaneously analyze their expression in failing hearts. METHODS: We analyzed left ventricular biopsies from 30 patients with heart failure undergoing heart transplantation and 12 organ donors. The mRNA levels of ACE, ACE2, chymase and endothelial nitric oxide synthase (eNOS), were quantified by real-time polymerase chain reaction and mast cell density was assessed by immunohistochemistry. The mRNA levels of the atrial natriuretic peptide (ANP) and the brain natriuretic peptide (BNP) were also quantified as controls. RESULTS: There was higher ACE and chymase mRNA expression and mast cell density in failing than in control myocardium and no changes in ACE2 expression were detected. eNOS mRNA levels were lower in failing hearts. Both ANP and BNP expression were higher in pathological than in control samples. CONCLUSIONS: These data document a decompensation of vasoactive systems that may contribute to the progressive impairment of the myocardial function in HF. On the other hand, ACE2 mRNA expression is not altered in human end-stage HF.

A new notch in the HIF belt: how hypoxia impacts differentiation.

In the current issue of Developmental Cell, work by Gustafsson and coworkers demonstrates that hypoxia synergizes with Notch to inhibit differentiation of myogenic and neural precursor cells. This effect requires a newly described interaction between the transcriptionally active form of HIF-1alpha and the intracellular domain of Notch.

Changes in lipoprotein lipase modulate tissue energy supply during stress.

We studied the variations caused by stress in lipoprotein lipase (LPL) activity, LPL-mRNA, and local blood flow in LPL-rich tissues in the rat. Stress was produced by body immobilization (Immo): the rat's limbs were taped to metal mounts, and its head was placed in a plastic tube. Chronic stress (2 h daily of Immo) decreased total LPL activity in mesenteric and epididymal white adipose tissue (WAT) and was accompanied by a weight reduction of these tissues. In limb muscle, heart, and adrenals, total LPL activity and mRNA levels increased, and, in plasma, LPL activity and mass also increased. Acute stress (30-min Immo) caused a decrease in total LPL activity only in retroperitoneal WAT and an increase in preheparin plasma active LPL, but the overall weight of this tissue did not vary significantly. We propose an early release of the enzyme from this tissue into the bloodstream by some unknown extracellular pathways or other local mechanisms. These changes in this key energy-regulating enzyme are probably induced by catecholamines. They modify the flow of energy substrates between tissues, switching the WAT from importer to exporter of free fatty acids and favoring the uptake by muscle of circulating triacylglycerides for energy supply. Moreover, we found that acute stress almost doubled blood flow in all WAT studied, favoring the export of free fatty acids.

Ascites from cirrhotic patients induces angiogenesis through the phosphoinositide 3-kinase/Akt signaling pathway.

BACKGROUND/AIMS: Ascites in patients with cirrhosis is associated with worsening of systemic hemodynamics. Thus, the aim of this study was to investigate the biological activity of ascites on endothelial cells. METHODS: Human umbilical vein endothelial cells (HUVECs) were used to investigate the angiogenic activity of ascites obtained from cirrhotic patients. RESULTS: Ascites-induced Akt activation, cell migration and tube formation in HUVECs. The pretreatment of HUVECs with the phosphatidylinositide 3-kinase (PI3-kinase) inhibitor LY294002, resulted in a decrease in chemotaxis and cell tube formation induced by ascites. Moreover, the inhibition of Akt activity in HUVECs by transduction of an inactive phosphorylation Akt mutant (AA-Akt), blocked tube formation. These angiogenic effects of ascites were also operative in vivo showing a PI3-kinase activation dependence in the angiogenesis induced by ascites. In addition, the preincubation of ascites with anti-fibronectin antibody led to a significant decrease in HUVECs migration, cell tube formation and in vivo angiogenesis. CONCLUSIONS: These results confirm the novel concept that ascites is a bioactive fluid which can modify vascular properties through the activation of the PI3-kinase/Akt pathway in endothelial cells. Furthermore, our results demonstrated that this ascites-induced mechanism is mediated, at least in part, by fibronectin.

Transduction of the liver with activated Akt normalizes portal pressure in cirrhotic rats.

BACKGROUND & AIMS: Portal hypertension in cirrhosis is secondary to an increase in hepatic resistance that occurs mainly through collagen deposition. However, recent evidence points to a major contribution by other factors, such as an intrahepatic reduction in nitric oxide production. Akt is a major activator of the endothelial nitric oxide synthase (eNOS) enzyme, but its potential role in intrahepatic resistance in cirrhosis is unknown. For this reason the aims of the present study were to determine whether there is an impaired Akt activation in cirrhotic livers and how this phenomenon relates to the decrease in NO production associated with portal hypertension. METHODS: Cirrhosis was induced in rats by carbon tetrachloride inhalation. Protein abundance and phosphorylation status of Akt and eNOS were examined by Western blotting. The role of Akt in the liver of cirrhotic rats was investigated through infection with adenoviruses encoding either beta-galactosidase (beta-gal) or constitutively active Akt (myr-Akt). RESULTS: The liver of cirrhotic animals showed a significant reduction in Akt and eNOS phosphorylation. Adenoviral delivery of myr-Akt restored eNOS phosphorylation and increased the intrahepatic concentration of guanosine 3',5'-cyclic monophosphate. These events were associated with normalization in portal pressure and a significant increase in mean arterial pressure after 3 days of adenoviral infection. In contrast, transduction of livers with beta-gal did not produce any change in these hemodynamic parameters. CONCLUSIONS: myr-Akt gene therapy restored Akt activation and NO production in the cirrhotic liver, suggesting that this therapy may be useful for the treatment of portal hypertension.

Effect of the V1a/V2-AVP receptor antagonist, Conivaptan, on renal water metabolism and systemic hemodynamics in rats with cirrhosis and ascites.

BACKGROUND: Selective V(2)-AVP receptor antagonists are effective in inducing aquaresis in humans and rats with cirrhosis, hyponatremia and water retention. However, it is unknown whether dual V(1a)/V(2)-AVP antagonists are also efficacious as aquaretic agents under these conditions. This is important, particularly considering that blockade of V(1a)-AVP receptors could aggravate cardiocirculatory function in decompensated cirrhosis. AIMS: To evaluate the renal, hormonal and hemodynamic effects induced by the chronic oral administration of the V(1a)/V(2)-AVP antagonist, Conivaptan, in rats with CCl(4)-induced cirrhosis, ascites and severe water retention. METHODS: We assessed the aquaretic efficacy of 10-day chronic oral administration of Conivaptan (0.5mg/kg body weight (bw)) in cirrhotic rats with hyponatremia and water retention. Urine volume (UV), osmolality (UOsm), and sodium excretion (U(Na)V) were measured daily. At the end of the study arterial pressure was also measured. RESULTS: Conivaptan produced an acute increase in UV, a reduction in UOsm and, at the end of the investigation, cirrhotic rats receiving the V(1a)/V(2)-AVP receptor antagonist did not show hyponatremia or hypoosmolality. Conivaptan also normalized U(Na)V without affecting creatinine clearance and arterial pressure. CONCLUSIONS: Dual V(1a)/V(2)-receptor antagonists may be therapeutically useful for the treatment of water retention and dilutional hyponatremia in human cirrhosis.

Nitric oxide synthase 3-dependent vascular remodeling and circulatory dysfunction in cirrhosis.

Vascular remodeling is an active process that consists in important modifications in the vessel wall. Endothelium-derived nitric oxide (NO) plays a major role in this phenomenon. We assessed wall thickness (WT), total wall area (TWA), lumen diameter, and total nuclei number/cross-section (TN) in cirrhotic rats with ascites and in control rats. A second group of cirrhotic rats received the NO synthesis inhibitor, L-NAME, or vehicle daily for 11 weeks and systemic hemodynamics, arterial compliance, aortic NO synthase 3 (NOS3) protein expression, and vascular morphology were analyzed. Cirrhotic vessels showed a significant reduction in WT, TWA, and TN as compared to control vessels. Long-term inhibition of NOS activity in cirrhotic rats resulted in a significant increase in WT, TWA, and TN as compared to cirrhotic rats receiving vehicle. NOS3 protein abundance was higher in aortic vessels of nontreated cirrhotic animals than in controls. This difference was abolished by chronic treatment with L-NAME. NOS inhibition in cirrhotic rats resulted in higher arterial pressure and peripheral resistance and lower arterial compliance than cirrhotic rats receiving vehicle. Therefore, vascular remodeling in cirrhosis with ascites is a generalized process with significant functional consequences that can be negatively modulated by long-term inhibition of NOS activity.

Hypoxia is an inducer of vasodilator agents in peritoneal macrophages of cirrhotic patients.

The aim of the investigation was to assess whether hypoxia induces the production of endogenous vasoactive peptides in macrophages of cirrhotic patients with ascites because low tissue oxygenation is a relatively frequent event in these patients. Peritoneal macrophages were isolated from ascites, seeded on well plates, and cultured at different times under hypoxic (5% O(2)) or normoxic conditions (21% O(2)). Then, accumulation of vasoactive peptides sensitive to hypoxia including endothelin-1 (ET-1), vascular endothelial growth factor (VEGF), and adrenomedullin (ADM) was measured. Only VEGF and ADM were constitutively secreted, and hypoxia further stimulated the release of these vasodilator peptides. In concordance, increased messenger RNA (mRNA) levels of VEGF and ADM were found at culturing macrophages in hypoxia. This characteristic response was not observed in circulating monocytes of either cirrhotic patients or healthy subjects. Next the expression of the transcription factor, hypoxia inducible factor 1 (HIF-1), was analyzed. Expression of HIF-1alpha and HIF-1beta messengers and HIF-1beta protein subunit remained unchanged regardless of O(2) tension, whereas HIF-1alpha protein subunit was overexpressed under hypoxic conditions. Moreover, conditioned medium from macrophages cultured under hypoxic conditions promoted a larger nitric oxide (NO) release in endothelial cells than that of normoxic macrophages. In conclusion, these data indicate that hypoxia induces the synthesis of VEGF and ADM in macrophages of cirrhotic patients, likely through HIF-1-enhanced transcriptional activity. These data suggest that a local reduction in O(2) tension could enhance the synthesis of macrophage-derived vasodilators, thus aggravating the circulatory disturbance of these patients.

Endogenous cannabinoids: a new system involved in the homeostasis of arterial pressure in experimental cirrhosis in the rat.

BACKGROUND & AIMS: Recent studies have described the existence of endogenous cannabinoids with vasodilator activity because of their interaction with peripheral CB1 receptors, anandamide being the most extensively investigated. The study investigated whether endogenous cannabinoids are involved in the pathogenesis of the cardiovascular disturbances in experimental cirrhosis. METHODS: Arterial pressure, cardiac output, and total peripheral resistance were measured before and after the administration of a cannabinoid CB1 receptor antagonist to cirrhotic rats with ascites and to control rats. Blood pressure was also assessed in normotensive recipient rats after the intravenous administration of blood cells or isolated monocytes obtained from cirrhotic and control rats. Moreover, the endogenous content of anandamide was measured in circulating monocytes of cirrhotic and control rats by gas chromatography/mass spectrometry. RESULTS: CB1 receptor blockade did not modify systemic hemodynamics in control rats, but significantly increased arterial pressure and peripheral resistance in cirrhotic animals. Blood cell suspension or monocytes from cirrhotic animals, but not from controls, induced arterial hypotension in recipient rats. Finally, anandamide was solely detected in monocytes of cirrhotic animals. CONCLUSIONS: Monocytes of cirrhotic rats with ascites are activated to produce anandamide and this substance contributes to arterial hypotension in experimental cirrhosis.