Tumor angiogenesis. Regulation of mural cell recruitment and endothelial sprouting

The formation of new blood vessels is an inherent part of many physiological and pathological processes. Currently, there is a strong hope pathological situations like stroke, diabetic retinopathy and cancer may be affected by pharmacological regulators of blood vessel formation. This thesis work has mainly been focused on two molecules implicated in blood vessel formation, platelet-derived growth factor (PDGF)-B and vascular endothelial growth factor (VEGF)-A. In vessels, PDGF-B is expressed by endothelial cells (EC) and act as a chemotactic and mitogenic signal via the PDGF receptor (PDGFR)-b on vascular smooth muscle cells (vSMC) and pericytes (PC). Tumor vessels are often abnormal and show a sparse association of vSMC/PC. We addressed the role of PDGF-B in vSMC/PC recruitment to tumor vessels using gain- and loss-of function analyses of PDGF-B. Our results show that vSMC/PC recruitment is dependent on PDGFRb and an EC source of PDGF-B. PDGF-B binds to the extracellular matrix in the close vicinity of EC through its retention motif. This spatial distribution of PDGF-B protein is needed for proper association of the PC with vessels; lack of PDGF-B retention leads to the extension of cellular processes away from the vessel, and to partial or complete PC detachment.VEGF-A stimulates migration and proliferation by binding to VEGF receptor expressed by EC. We have addressed the mechanism by which VEGF stimulate vessel formation by analyzing postnatal retinal vascularization. Our results revealed two functions of VEGF-A; guidance of highly specialized cells, tip-cells, at the tip of sprouting vessels, and proliferation of cells in the trailing cells, stalk-cells. Both these processes are mediated by VEGF receptor (VEGFR)-2. VEGF-A is produced in isoforms of 120, 164 or 188 amino acids where VEGF188 bind to the ECM, VEGF120 is soluble and VEGF164 has intermediate properties. Their spatial distribution creates a gradient sensed by filopodial extensions from the tip-cell. We also describe the existence of tip-cells with filopodia in tumors vessels. The extent of EC filopodia varies between tumors and correlates with vSMC/PC density and VEGF-A expression. Taken together, these results suggest that PDGF-B- and VEGF-A-stimulated processes in the vasculature are analogous. Migration of vSMC and EC seem to depend on the exact spatial distribution of PDGF-B and VEGF-A respectively. Proliferation of these cells on the other hand, appears to be regulated by the concentration of PDGF-B and VEGF-A. Our work also implies that anti-angiogenic therapy targeting VEGFR2 will inhibit the tip-cell and hence guided angiogenesis. The differences in sprouting phenotype in various tumors indicate that the angiogenic process of these tumors might differ. This, and variable vSMC/PC abundance in different tumors may implicate differences in responsiveness to anti-angiogenic therapy

No Association of LOXL1 Gene Polymorphisms with Alzheimer's Disease

Aggregation of amyloid-beta is one of the major characteristics in brains of patients with Alzheimer's disease (AD). Although several mechanisms behind the formation of such aggregates have been suggested the regulatory factors are still unknown. The present study aimed at investigating the association of lysyl oxidase-like 1 (LOXL1) polymorphisms with AD diagnosis and cerebrospinal fluid biomarkers (CSF) for the disease. Proteins of the lysyl oxidase (LOX) family are involved in cross-linking extracellular matrix proteins to insoluble fibers and have been associated with neurodegenerative diseases including AD. Genetic polymorphisms in LOXL1 (rs1048661, rs3825942, and rs2165241) have been linked to exfoliation syndrome and exfoliation glaucoma, conditions that have shown association with AD. The polymorphisms were genotyped by Taqman allelic discrimination in a study sample including AD patients (n = 318) and controls (n = 575). In a subgroup of the population, the polymorphisms were analyzed in relation to APOE epsilon 4 genotype and to CSF (T-tau, P-tau, and A beta(1-42)). No evidence for associations of these polymorphisms with risk for AD or any of the studied CSF biomarkers measured was found. These results do not support LOXL1 as being a major risk gene for AD

Amyloid precursor protein-b facilitates cell adhesion during early development in zebrafish

Understanding the biological function of amyloid beta (A beta) precursor protein (APP) beyond its role in Alzheimer's disease is emerging. Yet, its function during embryonic development is poorly understood. The zebrafish APP orthologue, Appb, is strongly expressed during early development but thus far has only been studied via morpholino-mediated knockdown. Zebrafish enables analysis of cellular processes in an ontogenic context, which is limited in many other vertebrates. We characterized zebrafish carrying a homozygous mutation that introduces a premature stop in exon 2 of the appb gene. We report that appb mutants are significantly smaller until 2 dpf and display perturbed enveloping layer (EVL) integrity and cell protrusions at the blastula stage. Moreover, appb mutants surviving beyond 48 hpf exhibited no behavioral defects at 6 dpf and developed into healthy and fertile adults. The expression of the app family member, appa, was also found to be altered in appb mutants. Taken together, we show that appb is involved in the initial development of zebrafish by supporting the integrity of the EVL, likely by mediating cell adhesion properties. The loss of Appb might then be compensated for by other app family members to maintain normal development

Complement Gene Single Nucleotide Polymorphisms and Biomarker Endophenotypes of Alzheimer\u27s Disease

The complement system has been implicated in both physiological synapse elimination and Alzheimer\u27s disease (AD). Here, we investigated associations between four single nucleotide polymorphisms (SNPs) in complement genes and cerebrospinal fluid (CSF) biomarkers for AD in 452 neurochemically or neuropathologically verified AD cases and 678 cognitively normal controls. None of the SNPs associated with risk of AD but there were potential associations of rs9332739 in the C2 gene and rs4151667 in the complement factor B gene with CSF tau levels (p = 0.023) and Mini-Mental State Examination scores (p = 0.012), both of which may be considered markers of disease intensity/severity

Repurposing proteasome inhibitors for improved treatment of triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is associated with poor prognosis and limited treatment options due to the lack of important receptors (estrogen receptor [ER], progesterone receptor [PR], and human epidermal growth factor receptor 2 [HER2]) used for targeted therapy. However, high-throughput in vitro drug screening of cell lines is a powerful tool for identifying effective drugs for a disease. Here, we determine the intrinsic chemosensitivity of TNBC cell lines to proteasome inhibitors (PIs), thereby identifying potentially potent 2-drug combinations for TNBC. Eight TNBC cell lines (BT-549, CAL-148, HCC1806, HCC38, HCC70, MDA-MB-436, MDA-MB-453, and MDA-MB-468) and two controls (MCF-10A and MCF-7) were first exposed to 18 drugs (11 PIs and 7 clinically relevant chemotherapeutic agents) as monotherapy, followed by prediction of potent 2-drug combinations using the IDACombo pipeline. The synergistic effects of the 2-drug combinations were evaluated with SynergyFinder in four TNBC cell lines (CAL-148, HCC1806, HCC38, and MDA-MB-468) and three controls (BT-474, MCF-7, and T47D) in vitro, followed by further evaluation of tumor regression in zebrafish tumor models established using HCC1806 and MCF-7 cells. Monotherapy identified nine effective drugs (bortezomib, carfilzomib, cisplatin, delanzomib, docetaxel, epoxomicin, MLN-2238, MLN-9708, and nedaplatin) across all cell lines. PIs (e.g., bortezomib, delanzomib, and epoxomicin) were highly potent drugs in TNBC cells, of which bortezomib and delanzomib inhibited the chymotrypsin-like activity of the 20 S proteasome by 100% at 10 µM. Moreover, several potent 2-drug combinations (e.g., bortezomib+nedaplatin and epoxomicin+epirubicin) that killed virtually 100% of cells were also identified. Although HCC1806- and MCF-7-derived xenografts treated with bortezomib+nedaplatin and carboplatin+paclitaxel were smaller, HCC1806 cells frequently metastasized to the trunk region. Taken together, we show that PIs used in combination with platinum agents or topoisomerase inhibitors exhibit increased efficiency with almost 100% inhibition in TNBC cell lines, indicating that PIs are therefore promising compounds to use as combination therapy for TNBC