Angiogenic Endothelial Cells Research Articles

Endoglin/CD105-Based Imaging of Cancer and Cardiovascular Diseases: A Systematic Review.

Molecular imaging of pathologic lesions can improve efficient detection of cancer and cardiovascular diseases. A shared pathophysiological feature is angiogenesis, the formation of new blood vessels. Endoglin (CD105) is a coreceptor for ligands of the Transforming Growth Factor-β (TGF-β) family and is highly expressed on angiogenic endothelial cells. Therefore, endoglin-based imaging has been explored to visualize lesions of the aforementioned diseases. This systematic review highlights the progress in endoglin-based imaging of cancer, atherosclerosis, myocardial infarction, and aortic aneurysm, focusing on positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), near-infrared fluorescence (NIRF) imaging, and ultrasound imaging. PubMed was searched combining the following subjects and their respective synonyms or relevant subterms: “Endoglin”, “Imaging/Image-guided surgery”. In total, 59 papers were found eligible to be included: 58 reporting about preclinical animal or in vitro models and one ex vivo study in human organs. In addition to exact data extraction of imaging modality type, tumor or cardiovascular disease model, and tracer (class), outcomes were described via a narrative synthesis. Collectively, the data identify endoglin as a suitable target for intraoperative and diagnostic imaging of the neovasculature in tumors, whereas for cardiovascular diseases, the evidence remains scarce but promising.

Rho GTPases in Retinal Vascular Diseases.

The Rho family of small GTPases (Rho GTPases) act as molecular switches that transduce extrinsic stimuli into cytoskeletal rearrangements. In vascular endothelial cells (ECs), Cdc42, Rac1, and RhoA control cell migration and cell–cell junctions downstream of angiogenic and inflammatory cytokines, thereby regulating vascular formation and permeability. While these Rho GTPases are broadly expressed in various types of cells, RhoJ is enriched in angiogenic ECs. Semaphorin 3E (Sema3E) releases RhoJ from the intracellular domain of PlexinD1, by which RhoJ induces actin depolymerization through competition with Cdc42 for their common effector proteins. RhoJ further mediates the Sema3E-induced association of PlexinD1 with vascular endothelial growth factor receptor (VEGFR) 2 and the activation of p38. Upon stimulation with VEGF-A, RhoJ facilitates the formation of a holoreceptor complex comprising VEGFR2, PlexinD1, and neuropilin-1, leading to the prevention of VEGFR2 degradation and the maintenance of intracellular signal transduction. These pleiotropic roles of RhoJ are required for directional EC migration in retinal angiogenesis. This review highlights the latest insights regarding Rho GTPases in the field of vascular biology, as it will be informative to consider their potential as targets for the treatment of aberrant angiogenesis and hyperpermeability in retinal vascular diseases.

Impact of CD105 Flow-Cytometric Expression on Childhood B-Acute Lymphoblastic Leukemia

BackgroundCD105 (Endoglin) is a receptor of the transforming growth factor-Beta (TGF- β) superfamily. It is expressed in angiogenic endothelial cells and is considered a powerful marker of angiogenesis and a potential main player in the pathogenesis of vascular diseases as well as tumor progression. CD105 expression was correlated with poor prognosis in many types of solid malignancies, however, its influence on hematological neoplasms is still an area of interest.PurposeTo assess the flow-cytometric expression of CD105 in childhood B-acute lymphoblastic leukemia (B-ALL) and its relation to disease response after the induction chemotherapy.Subjects and MethodsEighty children newly diagnosed with B-ALL were screened for flow-cytometric expression of CD105 at time of diagnosis, then they were followed up to detect their response to induction therapy.ResultsCD105 was expressed in 41.2% of B-ALL patients. Higher expression of CD105 was observed in high and very high-risk groups. The multivariate analysis considered CD105 positivity as an independent prognostic marker for response to induction therapy. Values higher than 2.5 Specific fluorescence indices (SFIs) and 35% expression were sensitive predictors to induction failure.ConclusionCD105 can be considered as a potential prognostic marker for the detection of response to induction therapy in childhood B-ALL, and it can serve to optimize treatment decisions.

Abstract PO030: A novel integrin-targeted therapeutic agent for prostate cancer

Abstract Disintegrins are disulfide-rich peptides, many containing an Arg-Gly-Asp (RGD) motif. They hold significant translational potential as anti-cancer agents based on their high-affinity/high-specificity interaction with tumor integrins and desirable pharmacological attributes. The integrin-binding activity of disintegrins depends on a mobile 11-amino acid loop displaying the RGD tripeptide motif or some other tripeptide motif. They bind only to the activated conformation of integrins on motile cancer cells and angiogenic endothelial cells. We designed a sequence-engineered RGD-disintegrin, vicrostatin (VCN), that can be produced at ~200mg/L from cell lysates of a bacterial recombinant system. VCN, a monomer (MW=7146), inhibits tumor cell adhesion and dissemination, and angiogenic endothelial cell invasion and tube formation. Integrins are cell surface adhesion receptors (containing a and b chains) operating at the interface between the extracellular matrix and cytoskeletal apparatus. A sub-group of integrins recognize the RGD sequence present in key extracellular matrix proteins involved in prostate cancer (PC) growth and dissemination. Integrins are involved in bi-directional signaling interactions that alter cellular functions. Among these interactions are those involved in PC growth, metastasis and angiogenesis. Integrin avb3 plays an important role in the development of bone metastases, supporting our belief that it, as well as other RGD-integrins, are important therapeutic targets in advanced PC. We examined the effect of treatment of a xenograft model of human PC using a liposomal formulation of VCN (LVCN). PC3 cells were implanted subcutaneously in 5-week old male nude mice. LVCN and VCN (100µg VCN equivalent per dose) administration was initiated IV when tumors became palpable (14 days). Tumor size was measured weekly by caliper in a blinded study. There was a 75% reduction in tumor growth in LVCN-treated animals compared to controls. VCN displayed little anti-tumor activity most likely due to its lack of availability to the PC cells. We also examined the anti-angiogenic activity of LVCN in this model using CD31/PECAM immunohistochemistry and observed a significant decrease in tumor-associated microvessel density. We then evaluated therapeutic efficacy of LVCN in treating bone metastases. CWR22rV1 PC cells were suspended in a solution of matrigel and injected into a drilled hole in the proximal left tibia of immunodeficient mice (100,000 cells/10µL). Following injection, the diameters of both tibias were measured by caliper twice weekly. Since CWR22rV1 cells are androgen dependent, animals received daily injections of testosterone. Tumors grew slowly initially, but at 20 days tumors were evident and treatment began. LVCN and VCN were administered twice weekly via IV injection (100µg per dose) for 5 weeks. LVCN displayed ~81% inhibition of tumor growth compared to VCN, PBS or empty liposomes. Our findings provide strong support for the effectiveness of LVCN as an inhibitor of PC growth and metastases. Citation Format: Francis S. Markland, Stephen Swenson, Radu Minea, Jacek Pinski. A novel integrin-targeted therapeutic agent for prostate cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO030.

Mechanistic modeling of vascular tumor growth: an extension of Biot’s theory to hierarchical bi-compartment porous medium systems

Existing continuum multiphase tumor growth models typically do not include microvasculature, or if present, this is modeled as a non-deformable network of vessels. Vasculature behavior and blood flow are usually non-coupled with the underlying tumor phenomenology from the mechanical viewpoint; hence, phenomena like vessel compression/occlusion modifying microcirculation and oxygen supply cannot be taken into account. Here, the tumor tissue is modeled as a reactive bi-compartment porous medium: the extracellular matrix constitutes the solid scaffold; blood flows in the vascular porosity, whereas the extravascular porous compartment is saturated by two cell phases and interstitial fluid (mixture of water and nutrient species). The pressure difference between blood and the extravascular overall pressure is sustained by vessel walls and drives shrinkage or dilatation of the vascular porosity. Model closure is achieved thanks to a consistent non-conventional definition of the Biot’s effective stress tensor. Angiogenesis is modeled by introducing a vascularization state variable and accounting for tumor angiogenic factors and endothelial cells. Closure relationships and mass exchange terms related to vessel formation are detailed in a numerical example reproducing the principal features of angiogenesis. This example is preceded by a first pedagogical numerical study on one-dimensional bio-consolidation. Results demonstrate that the bi-compartment poromechanical model is fully coupled (the external loads impact fluid flow in both porous compartments) and that it can serve as a basis for further applications like modeling of drug delivery and tissue ulceration.

Peripheral sensory neurons promote angiogenesis in neurovascular models derived from hESCs.

In the adult tissues, blood vessels traverse the body with neurons side by side; and share common signaling molecules. Developmental studies on animal models have shown that peripheral sensory neurons (PSNs) secrete angiogenic factors and endothelial cells (ECs) secrete neurotrophic factors which contribute to their coexistence, thereby forming the peripheral neurovascular (PNV) unit. Despite the large number of studies showing that innervation and vascularization complement each other, the interaction between human PSNs and ECs is still largely unknown. To study this interaction and to evaluate if PSNs affect angiogenesis, we derived both PSNs and ECs from human embryonic stem cells (hESCs) and developed a co-culture system. Seeding the two cell types together showed that PSNs induced endothelial morphogenesis with formation of vessel-like structures (VLSs). The PSN precursors, neural crest stem cells also induced VLS formation in the co-culture system; however, to a lesser extent. This sheds new light on the in vitro angiogenic potential of these cell types. PSNs derived from hESCs are powerful tools for studying development and disease as human PSNs are inaccessible for in vitro assays. Our novel approach, with optimized media condition allowed for integrating hESC-derived PSNs with hESC-derived ECs in three-dimensional (3D) collagen gel for creating a completely humanised PNV model. This preliminary model showed that innervation improves the development of vascularized channels in vitro, and provides insight to the development of innervated 3D models in future.

Simultaneously targeting cancer-associated fibroblasts and angiogenic vessel as a treatment for TNBC.

Fibrotic tumor stroma plays an important role in facilitating triple-negative breast cancer (TNBC) progression and chemotherapeutic resistance. We previously reported a rationally designed protein (ProAgio) that targets integrin αvβ3 at a novel site. ProAgio induces apoptosis via the integrin. Cancer-associated fibroblasts (CAFs) and angiogenic endothelial cells (aECs) in TNBC tumor express high levels of integrin αvβ3. ProAgio effectively induces apoptosis in CAFs and aECs. The depletion of CAFs by ProAgio reduces intratumoral collagen and decreases growth factors released from CAFs in the tumor, resulting in decreased cancer cell proliferation and apoptotic resistance. ProAgio also eliminates leaky tumor angiogenic vessels, which consequently reduces tumor hypoxia and improves drug delivery. The depletion of CAFs and reduction in hypoxia by ProAgio decreases lysyl oxidase (LOX) secretion, which may play a role in the reduction of metastasis. ProAgio stand-alone or in combination with a chemotherapeutic agent provides survival benefit in TNBC murine models, highlighting the therapeutic potential of ProAgio as a treatment strategy.

Sulfated non-anticoagulant low molecular weight heparin in the prevention of cancer and non-cancer associated thrombosis without compromising hemostasis

IntroductionCancer-associated thrombosis (CAT) accounts for about 20% of all cases of Venous Thromboembolism (VTE). Tissue factor (TF) is documented to be highly expressed on cancer cells and pathological angiogenic endothelial cells. Here, we used a novel oxidized sulfated ultra-LMWH, S-NACH, which is devoid of anti-factor Xa and IIa activities with limited to no systemic anticoagulant effects. This sulfated form has enhanced binding to vascular endothelial cells (EC) and releases and potentiates the action of tissue factor pathway inhibitor (TFPI). S-NACH binds with high affinity to EC, releases and binds to EC TFPI, and promotes vascular antithrombotic effect with limited to no risk of bleeding complications. Materials and methodsWe investigated the effects of S-NACH on clot kinetics in vitro and in vivo. Also, we investigated the effects of S-NACH on CAT mediated by human acute leukemia cells (K562) and human pancreatic cancer cells (SUIT2). ResultsS-NACH was associated with ~3-fold increase of TFPI 2 levels within 3 h. Also, S-NACH reversed the hypercoagulability state that is associated with cancer cells in vitro. In vivo, S-NACH at 20 mg/kg subcutaneously (SC) had no effect on bleeding time compared to both tinzaparin and enoxaparin at 5 mg/kg SC. S-NACH did not show any anti-IIa or anti-Xa activities in comparison to tinzaparin and enoxaparin (p < 0.001). ConclusionData suggest the importance of S-NACH through its EC binding, EC TFPI release and its interaction with TFPI in enhancing its activity in the prevention of cancer and non-cancer associated thrombosis with limited to no bleeding complications.

MigrationStimulating Factor (MSF): Its Role in the Tumour Microenvironment.

Migration Stimulating Factor(MSF) is a 70kDa truncated isoform of fibronectin (FN); its mRNA is generated from the FN gene by an unusual two-stage processing. Unlike full-length FN, MSF is not a matrix molecule but a soluble protein which displays cytokine-like activities not displayed by any other FN isoform due to steric hindrance. There are two isoforms of MSF; these are referred to as MSF+aa and MSF-aa, while the term MSF is used to include both.MSF was first identified as a motogen secreted by foetal and cancer-associated fibroblasts in tissue culture. It is also produced by sprouting (angiogenic) endothelial cells, tumour cells and activated macrophages. Keratinocytes and resting endothelial cells secrete inhibitors of MSF that have been identified as NGAL and IGFBP-7, respectively. MSF+aa and MSF-aa show distinct functionality in that only MSF+aa is inhibited by NGAL.MSF is present in 70-80% of all tumours examined, expressed by the tumour cells as well as by fibroblasts, endothelial cells and macrophages in the tumour microenvironment (TME). High MSF expression is associated with tumour progression and poor prognosis in all tumours examined, including breast carcinomas, non-small cell lung cancer (NSCLC), salivary gland tumours (SGT) and oral squamous cell carcinomas (OSCC). Epithelial and stromal MSF carry independent prognostic value. MSF is also expressed systemically in cancer patients, being detected in serum and produced by fibroblast from distal uninvolved skin. MSF-aa is the main isoform associated with cancer, whereas MSF+aa may be expressed by both normal and malignant tissues.The expression of MSF is not invariant; it may be switched on and off in a reversible manner, which requires precise interactions between soluble factors present in the TME and the extracellular matrix in contact with the cells. MSF expression in fibroblasts may be switched on by a transient exposure to several molecules, including TGFβ1 and MSF itself, indicating an auto-inductive capacity.Acting by both paracrine and autocrine mechanisms, MSF stimulates cell migration/invasion, induces angiogenesis and cell differentiation and alters the matrix and cellular composition of the TME. MSF is also a survival factor for sprouting endothelial cells. IGD tri- and tetra-peptides mimic the motogenic and angiogenic activities of MSF, with both molecules inhibiting AKT activity and requiring αvβ3 functionality. MSF is active at unprecedently low concentrations in a manner which is target cell specific. Thus, different bioactive motifs and extracellular matrix requirements apply to fibroblasts, endothelial cells and tumour cells. Unlike other motogenic and angiogenic factors, MSF does not affect cell proliferation but it stimulates tumour growth through its angiogenic effect and downstream mechanisms.The epithelial-stromal pattern of expression and range of bioactivities displayed puts MSF in the unique position of potentially promoting tumour progression from both the "seed" and the "soil" perspectives.

Abstract 13610: Endothelial Cu Transporter Atp7a Promotes Vegfr2 Signaling and Post-ischemic Neovascularization via Regulating Autophagy

Background: VEGFR2 (KDR/Flk1) signaling in endothelial cells (ECs) plays a central role in angiogenesis. Copper (Cu) is essential micronutrient and has been implicated in angiogenesis. The P-type ATPase transporter ATP7A is key regulator of Cu homeostasis but its role in VEGFR2 signaling in ECs and post-ischemic neovascularization is entirely unknown. Results: Here we show that ATP7A expression was dramatically increased in the angiogenic ECs in mice hindlimb ischemia model. EC-specific ATP7A deficient mice or Cu transporter-dysfunctional ATP7A mut mice showed significant decrease in blood flow recovery and CD31+ capillary density (angiogenesis) in ischemic tissues compared to their control mice. In cultured human ECs, ATP7A knockdown with siRNA significantly inhibited VEGF-induced migration (67%), capillary network formation on Matrigel (46%). Immunofluorescence, co-immunoprecipitation, and proximity ligation assays showed that VEGF stimulated ATP7A translocation from the trans-Golgi network to the plasma membrane where it bound to VEGFR2. Surprisingly, loss of ATP7A promoted VEGF-induced VEGFR2 protein degradation (56%) and inhibited VEGFR2 signaling via enhancing VEGFR2 ubiquitination (2.0-fold) in a Cu-independent manner. This was associated with reduced cell surface VEGFR2 expression, increased VEGFR2 binding to selective autophagic cargo/adaptor p62/SQSTM1 (60%) and LC3-GFP+autophagic puncta (34%) and autolysosome formation (transmission electron microscopy). Inhibition of autophagy by bafilomycin A1 or chloroquine prevented enhanced VEGFR2 degradation in ATP7A-depleted ECs. Furthermore, overexpression of p62, but not p62 lacking ubiquitin binding domain, or autophagy inducer rapamycin treatment increased VEGFR2 degradation (26% and 87%, respectively) and p62 overexpression inhibited ECs migration (29%). Enhanced autophagy flux due to ATP7A dysfunction in vivo was confirmed by employing autophagy reporter CAG-ATP7A mut -RFP-EGFP-LC3 transgenic mice. Conclusion: Our study uncovers the unexpected and novel function of ATP7A to limit autophagic degradation of VEGFR2, thereby promoting VEGFR2 signaling and angiogenesis, which restores neovascularization in ischemic disease.

Abstract 14078: Ros-dependent Sumoylation of Cu Chaperone Atox1 Drives Its Nuclear Translocation to Promote Inflammatory Angiogenesis Induced by Ischemic Injury

Introduction: Neovascularization in response to ischemia depends on inflammation, angiogenesis and reactive oxygen species (ROS). Copper (Cu) is implicated in inflammation and angiogenesis. We reported that cytosolic Cu chaperone Atox1 activates secretory Cu enzymes lysyl oxidase (LOX), while nuclear Atox1 functions as a Cu-dependent transcription factor to promote ROS/NFkB-dependent inflammation in endothelial cells (ECs). However, mechanism of Atox1 nuclear translocation as well as role of endothelial Atox1 in inflammatory angiogenesis in vivo remain unknown. SUMOylation and its deSUMOylation by SENPs regulates transcription factor function. Silica analysis identified a conserved putative SUMOylation motif at Lys(K3) of Atox1. Results: Atox1 expression was dramatically increased in angiogenic ECs in mice hindlimb ischemia model. EC-specific Atox1-deficient mice significantly reduced angiogenesis (CD31+, 67%) and Mac+ inflammatory cells in ischemic tissues. In cultured ECs, inflammatory cytokine TNFα or hypoxia promoted Atox1 nuclear translocation and Atox1 SUMOylation (3.6-fold), which were inhibited by antioxidant NAC or overexpression of “SUMO-dead” Atox1K3R. Mechanistically, TNFα induced Cys603 oxidation/inactivation of SENP1 in cytosol, which in turn increased Atox1 SUMOylation and nuclear translocation. Functionally, siAtox1or Atox1K3R inhibited TNFα-induced inflammatory/angiogenic genes VCAM/ICAM, IL-15 and RANTES. In nucleus with reduced state, ChIP assay using SUMO-Atox1 revealed that Atox1 deSUMOylation by nuclear SENP1 increases Atox1 transcriptional activity for inflammatory genes. In parallel, Atox1K3R which maintains Cu chaperone function inhibited TNFα-induced EC permeability by activating LOX. In vivo, Atox1 SUMOylation was increased after hindlimb ischemia while CRISPR/Cas9-generated SUMO-dead Atox1K3R knock-in mice showed impaired angiogenesis in hindlimb ischemia model. Conclusion: Atox1 SUMOylation via oxidative/inactivation of SENP1 in cytosol promotes: 1) its translocation to nucleus where deSUMOylated Atox1 can function as Cu-dependent transcription factor to drive inflammatory angiogenesis and 2) EC barrier dysfunction in inflamed/hypoxic ECs after ischemic injury.

Multiscale Selectivity and in vivo Biodistribution of NRP-1-Targeted Theranostic AGuIX Nanoparticles for PDT of Glioblastoma.

BackgroundLocal recurrences of glioblastoma (GBM) after heavy standard treatments remain frequent and lead to a poor prognostic. Major challenges are the infiltrative part of the tumor tissue which is the ultimate cause of recurrence. The therapeutic arsenal faces the difficulty of eradicating this infiltrating part of the tumor tissue while increasing the targeting of tumor and endogenous stromal cells such as angiogenic endothelial cells. In this aim, neuropilin-1 (NRP-1), a transmembrane receptor mainly overexpressed by endothelial cells of the tumor vascular system and associated with malignancy, proliferation and migration of GBM, highlighted to be a relevant molecular target to promote the anti-vascular effect of photodynamic therapy (VTP).MethodsThe multiscale selectivity was investigated for KDKPPR peptide moiety targeting NRP-1 and a porphyrin molecule as photosensitizer (PS), both grafted onto original AGuIX design nanoparticle. AGuIX nanoparticle, currently in Phase II clinical trials for the treatment of brain metastases with radiotherapy, allows to achieve a real-time magnetic resonance imaging (MRI) and an accumulation in the tumor area by EPR (enhanced permeability and retention) effect. Using surface-plasmon resonance (SPR), we evaluated the affinities of KDKPPR and scramble free peptides, and also peptides-conjugated AGuIX nanoparticles to recombinant rat and human NRP-1 proteins. For in vivo selectivity, we used a cranial window model and parametric maps obtained from T2*-weighted perfusion MRI analysis.ResultsThe photophysical characteristics of the PS and KDKPPR molecular affinity for recombinant human NRP-1 proteins were maintained after the functionalization of AGuIX nanoparticle with a dissociation constant of 4.7 μM determined by SPR assays. Cranial window model and parametric maps, both revealed a prolonged retention in the vascular system of human xenotransplanted GBM. Thanks to the fluorescence of porphyrin by non-invasive imaging and the concentration of gadolinium evaluated after extraction of organs, we checked the absence of nanoparticle in the brains of tumor-free animals and highlighted elimination by renal excretion and hepatic metabolism.ConclusionPost-VTP follow-ups demonstrated promising tumor responses with a prolonged delay in tumor growth accompanied by a decrease in tumor metabolism.

Slug regulates the Dll4-Notch-VEGFR2 axis to control endothelial cell activation and angiogenesis

Slug (SNAI2), a member of the well-conserved Snail family of transcription factors, has multiple developmental roles, including in epithelial-to-mesenchymal transition (EMT). Here, we show that Slug is critical for the pathological angiogenesis needed to sustain tumor growth, and transiently necessary for normal developmental angiogenesis. We find that Slug upregulation in angiogenic endothelial cells (EC) regulates an EMT-like suite of target genes, and suppresses Dll4-Notch signaling thereby promoting VEGFR2 expression. Both EC-specific Slug re-expression and reduced Notch signaling, either by γ-secretase inhibition or loss of Dll4, rescue retinal angiogenesis in SlugKO mice. Conversely, inhibition of VEGF signaling prevents excessive angiogenic sprouting of Slug overexpressing EC. Finally, endothelial Slug (but not Snail) is activated by the pro-angiogenic factor SDF1α via its canonical receptor CXCR4 and the MAP kinase ERK5. Altogether, our data support a critical role for Slug in determining the angiogenic response during development and disease.

Single-nucleus transcriptome analysis reveals dysregulation of angiogenic endothelial cells and neuroprotective glia in Alzheimer’s disease

Alzheimer's disease (AD) is the most common form of dementia but has no effective treatment. A comprehensive investigation of cell type-specific responses and cellular heterogeneity in AD is required to provide precise molecular and cellular targets for therapeutic development. Accordingly, we perform single-nucleus transcriptome analysis of 169,496 nuclei from the prefrontal cortical samples of AD patients and normal control (NC) subjects. Differential analysis shows that the cell type-specific transcriptomic changes in AD are associated with the disruption of biological processes including angiogenesis, immune activation, synaptic signaling, and myelination. Subcluster analysis reveals that compared to NC brains, AD brains contain fewer neuroprotective astrocytes and oligodendrocytes. Importantly, our findings show that a subpopulation of angiogenic endothelial cells is induced in the brain in patients with AD. These angiogenic endothelial cells exhibit increased expression of angiogenic growth factors and their receptors (i.e., EGFL7, FLT1, and VWF) and antigen-presentation machinery (i.e., B2M and HLA-E). This suggests that these endothelial cells contribute to angiogenesis and immune response in AD pathogenesis. Thus, our comprehensive molecular profiling of brain samples from patients with AD reveals previously unknown molecular changes as well as cellular targets that potentially underlie the functional dysregulation of endothelial cells, astrocytes, and oligodendrocytes in AD, providing important insights for therapeutic development.

Modulation of Cancer-Associated Fibrotic Stroma by An Integrin αvβ3 Targeting Protein for Pancreatic Cancer Treatment.

Background & AimsPancreatic ductal adenocarcinoma (PDAC) is resistant to most therapeutics owing to dense fibrotic stroma orchestrated by cancer-associated pancreatic stellate cells (CAPaSC). CAPaSC also support cancer cell growth, metastasis, and resistance to apoptosis. Currently, there is no effective therapy for PDAC that specifically targets CAPaSC. We previously reported a rationally designed protein, ProAgio, that targets integrin αvβ3 at a novel site and induces apoptosis in integrin αvβ3-expressing cells. Because both CAPaSC and angiogenic endothelial cells express high levels of integrin αvβ3, we aimed to analyze the effects of ProAgio in PDAC tumor.MethodsExpression of integrin αvβ3 was examined in both patient tissue and cultured cells. The effects of ProAgio on CAPaSC were analyzed using an apoptosis assay kit. The effects of ProAgio in PDAC tumor were studied in 3 murine tumor models: subcutaneous xenograft, genetic engineered (KrasG12D; p53R172H; Pdx1-Cre, GEM-KPC) mice, and an orthotopic KrasG12D; p53R172H; Pdx1-Cre (KPC) model.ResultsProAgio induces apoptosis in CAPaSC. ProAgio treatment significantly prolonged survival of a genetically engineered mouse–KPC and orthotopic KPC mice alone or in combination with gemcitabine (Gem). ProAgio specifically induced apoptosis in CAPaSC, resorbed collagen, and opened collapsed tumor vessels without an increase in angiogenesis in PDAC tumor, enabling drug delivery into the tumor. ProAgio decreased intratumoral insulin-like growth factor 1 levels as a result of depletion of CAPaSC and consequently decreased cytidine deaminase, a Gem metabolism enzyme in cancer cells, and thereby reduced resistance to Gem-induced apoptosis.ConclusionsOur study suggests that ProAgio is an effective PDAC treatment agent because it specifically depletes CAPaSC and eliminates tumor angiogenesis, thereby enhancing drug delivery and Gem efficacy in PDAC tumors.

Laminin-511 and α6 integrins regulate the expression of CXCR4 to promote endothelial morphogenesis.

During angiogenesis, endothelial cells engage components of the extracellular matrix through integrin-mediated adhesion. Endothelial expression of laminin-411 and laminin-511 is known to promote vessel stability. However, little is known about the contribution of these laminins to endothelial morphogenesis. We used two organotypic cell culture angiogenesis assays, in conjunction with RNAi approaches, to demonstrate that depletion of either the α4 chain of laminin-411 (LAMA4) or the α5 chain of laminin-511 (LAMA5) from endothelial cells inhibits sprouting and tube formation. Depletion of α6 (ITGA6) integrins resulted in similar phenotypes. Gene expression analysis indicated that loss of either laminin-511 or α6 integrins inhibited the expression of CXCR4, a gene previously associated with angiogenic endothelial cells. Pharmacological or RNAi-dependent inhibition of CXCR4 suppressed endothelial sprouting and morphogenesis. Importantly, expression of recombinant CXCR4 rescued endothelial morphogenesis when α6 integrin expression was inhibited. Additionally, the depletion of α6 integrins from established tubes resulted in the loss of tube integrity and laminin-511. Taken together, our results indicate that α6 integrins and laminin-511 can promote endothelial morphogenesis by regulating the expression of CXCR4 and suggest that the α6-dependent deposition of laminin-511 protects the integrity of established endothelial tubes.

NGRKC16-lipopeptide assisted liposomal-withaferin delivery for efficient killing of CD13 receptor-expressing pancreatic cancer and angiogenic endothelial cells

Cancer treatment necessitates targeted and efficient drug delivery to reduce drug-mediated adverse collateral effects. We develop herein a new liposomal delivery system which targets both CD13 receptor-positive cancer epithelial and angiogenic endothelial cells using the homing peptide (NGR), as liposome surface bound targeting ligand. The ligand NGR, upon covalent conjugation to a C16-aliphatic twin-chain lipid via a lysine (K) spacer, afforded C16-lipopeptide. Importantly, the circulation stable liposomes of NGRKC16-lipopeptide containing encapsulated withaferin-A (WFA), a multipotent drug, exhibited receptor-mediated uptake and apoptosis-mediated cell death in both CD13 receptor-positive pancreatic cancer cells and angiogenic endothelial cells, while the other non-targeting control formulation failed to do so. In a Chick embryo angiogenesis assay, the targeted formulation selectively inhibited angiogenesis, i.e., sprouting of new blood vessels from the pre-existing blood vessels. These finding revealed therapeutic potential of WFA-encapsulated liposomal formulation of NGRKC16-lipopeptide for simultaneous killing of CD13-positive pancreatic cancer as well as angiogenic endothelial cells. As angiogenesis, endorsed by endothelial cells and aggressiveness in tumor epithelial cells are symbiotically related, the presently described CD13 receptor selective WFA-encapsulated liposomal formulation of NGRKC16-lipopeptide may find future use for the treatment of aggressive pancreatic cancer.

Caveolin-1 regulates the ASMase/ceramide-mediated radiation response of endothelial cells in the context of tumor\u2013stroma interactions

The integral membrane protein caveolin-1 (CAV1) plays a central role in radioresistance-mediating tumor–stroma interactions of advanced prostate cancer (PCa). Among the tumor–stroma, endothelial cells (EC) evolved as critical determinants of the radiation response. CAV1 deficiency in angiogenic EC was already shown to account for increased apoptosis rates of irradiated EC. This study explores the potential impact of differential CAV1 levels in EC on the acid sphingomyelinase (ASMase)/ceramide pathway as a key player in the regulation of EC apoptosis upon irradiation and cancer cell radioresistance. Enhanced apoptosis sensitivity of CAV1-deficient EC was associated with increased ASMase activity, ceramide generation, formation of large lipid platforms, and finally an altered p38 mitogen-activated protein kinase (MAPK)/heat-shock protein 27 (HSP27)/AKT (protein kinase B, PKB) signaling. CAV1-deficient EC increased the growth delay of LNCaP and PC3 PCa cells upon radiation treatment in direct 3D spheroid co-cultures. Exogenous C6 and C16 ceramide treatment in parallel increased the growth delay of PCa spheroids and induced PCa cell apoptosis. Analysis of the respective ceramide species in PCa cells with increased CAV1 levels like those typically found in radio-resistant advanced prostate tumors further revealed an upregulation of unsaturated C24:1 ceramide that might scavenge the effects of EC-derived apoptosis-inducing C16 ceramide. Higher ASMase as well as ceramide levels could be confirmed by immunohistochemistry in human advanced prostate cancer specimen bearing characteristic CAV1 tumor–stroma alterations. Conclusively, CAV1 critically regulates the generation of ceramide-dependent (re-)organization of the plasma membrane that in turn affects the radiation response of EC and adjacent PCa cells. Understanding the CAV1-dependent crosstalk between tumor cells and the host-derived tumor microvasculature and its impact on radiosensitivity may allow to define a rational strategy for overcoming tumor radiation resistance improving clinical outcomes by targeting CAV1.

Viral Therapy for Glioblastoma

Viral Therapy for Glioblastoma

Microchannels Are an Architectural Cue That Promotes Integration and Vascularization of Silk Biomaterials in Vivo.

Functional integration of implanted biomaterials and bioengineered tissues in vivo requires effective and timely vascular ingrowth. While many vascularization strategies rely on delivery of angiogenic growth factors or endothelial cells to promote vascular ingrowth, the effect of physical and architectural features of biomaterials on the vascularization process is less well understood. Microchannels are a simple, accessible architectural feature frequently engineered into 3D biomaterials to promote mass transfer. In this study, the effect of microchannels on the integration and vascularization of 3D porous silk scaffolds was explored over a 14 week period. An array of 508 μm diameter microchannels spanning the length of critically sized, porous silk scaffolds significantly improved tissue ingrowth into the constructs. At week 6, all silk scaffolds (n = 8) with microchannels showed complete tissue infiltration throughout the construct, while only one of eight (12.5%) did so in the absence of microchannels. The presence of microchannels improved silk scaffold vascularization with significantly more vessels per unit area in the presence of microchannels. The vessel size distribution was similar in both scaffold types, but a shift in distribution toward smaller vessels was observed in the presence of microchannels. The blood vessels in silk scaffolds were perfused, functional and connected to the animal's cardiovascular system, as demonstrated by the presence of red blood cells in the vessel lumens, and effective delivery of a contrast agent the vessels inside the scaffold. This study demonstrates the utility of microchannels as a simple architectural feature that significantly improves vascularization and integration of implanted biomaterials.