Secretion of Proteins and Antibody Fragments From Transiently Transfected Endothelial Progenitor Cells

In neurodegenerative diseases such as Alzheimer\u27s disease, Parkinson\u27s disease, multiple sclerosis and amyotrophic lateral sclerosis, neuroinflammation can lead to blood-brain barrier (BBB) breakdown. After intravenous or intra-arterial injection into mice, endothelial progenitor cells (EPCs) home to the damaged BBB to promote neurovascular repair. Autologous EPCs transfected to express specific therapeutic proteins offer an innovative therapeutic option. Here, we demonstrate that EPC transfection by electroporation with plasmids encoding the reporter protein GFP or an anti-beta-amyloid antibody fragment (Fab) leads to secretion of each protein. We also demonstrate the secreted anti-beta-amyloid Fab protein functions in beta-amyloid aggregate solubilization

Secretion of proteins and antibody fragments from transiently transfected endothelial progenitor cells

In neurodegenerative diseases such as Alzheimer\u27s disease, Parkinson\u27s disease, multiple sclerosis and amyotrophic lateral sclerosis, neuroinflammation can lead to blood-brain barrier (BBB) breakdown. After intravenous or intra-arterial injection into mice, endothelial progenitor cells (EPCs) home to the damaged BBB to promote neurovascular repair. Autologous EPCs transfected to express specific therapeutic proteins offer an innovative therapeutic option. Here, we demonstrate that EPC transfection by electroporation with plasmids encoding the reporter protein GFP or an anti-beta-amyloid antibody fragment (Fab) leads to secretion of each protein. We also demonstrate the secreted anti-beta-amyloid Fab protein functions in beta-amyloid aggregate solubilization

Suppression of hypoxia-induced HIF-1α and of angiogenesis in endothelial cells by myo-inositol trispyrophosphate-treated erythrocytes

Allosteric regulation of oxygen delivery by RBCs may have significant effects on tumor growth. Indeed, angiogenesis, the formation of new blood vessels, is induced in growing tumors by low oxygen partial pressure. Hypoxia-inducible genes are switched on, among which are the VEGF gene and its receptors. Most important, under hypoxia, hypoxia-inducible factor 1α has a significantly prolonged half-life and up-regulates a number of hypoxia genes. Human microvascular endothelial cells (MECs), when subjected in vitro to hypoxia, align to form vessel-like structures as in the angiogenic process. We report here that, when cultured in hypoxic conditions in the presence of human RBCs loaded with a new membrane-permeant allosteric effector of Hb, myo-inositol trispyrophosphate (ITPP), endothelial cells (ECs) do not align, i.e., do not form “vessel”-like structures, because the “loaded” RBCs are capable of releasing under hypoxia more oxygen than their “normal” counterparts. Levels of VEGF and of hypoxia-inducible factor 1α, elevated in the human MECs under hypoxia, were dramatically reduced or even suppressed in the presence of the ITPP-loaded RBCs. Treatment of these ECs directly with free ITPP at different concentrations had no effect on their ability to undertake angiogenesis. Incubation with ITPP enhances the capacity of Hb to release bound oxygen, leading to higher oxygen tension in the hypoxic environment, thus inhibiting hypoxia-induced angiogenesis. These observations are suggestive of a potential in vivo role of ITPP-loaded, “low-O(2)-affinity” RBCs in cancer therapy

“Endothelial Antibody Factory” at the Blood Brain Barrier: Novel Approach to Therapy of Neurodegenerative Diseases

The failures of anti-β-amyloid immunotherapies suggested that the very low fraction of injected antibodies reaching the brain parenchyma due to the filtering effect of the BBB may be a reason for the lack of therapeutic effect. However, there is no treatment, as yet, for the amyotrophic lateral sclerosis (ALS) despite substantial evidence existing of the involvement of TDP-43 protein in the evolution of ALS. To circumvent this filtering effect, we have developed a novel approach to facilitate the penetration of antibody fragments (Fabs) into the brain parenchyma. Leveraging the homing properties of endothelial progenitor cells (EPCs), we transfected, ex vivo, such cells with vectors encoding anti-β-amyloid and anti-TDP43 Fabs turning them into an “antibody fragment factory”. When injected these cells integrate into the BBB, where they secrete anti-TDP43 Fabs. The results showed the formation of tight junctions between the injected engineered EPCs and the unlabeled resident endothelial cells. When the EPCs were further modified to express the anti-TDP43 Fab, we could observe integration of these cells into the vasculature and the secretion of Fabs. Results confirm that production and secretion of Fabs at the BBB level leads to their migration to the brain parenchyma where they might exert a therapeutic effect

Enhanced exercise capacity in mice with severe heart failure treated with an allosteric effector of hemoglobin, myo-inositol trispyrophosphate

A major determinant of maximal exercise capacity is the delivery of oxygen to exercising muscles. myo-Inositol trispyrophosphate (ITPP) is a recently identified membrane-permeant molecule that causes allosteric regulation of Hb oxygen binding affinity. In normal mice, i.p. administration of ITPP (0.5–3 g/kg) caused a dose-related increase in the oxygen tension at which Hb is 50% saturated (p50), with a maximal increase of 31%. In parallel experiments, ITPP caused a dose-related increase in maximal exercise capacity, with a maximal increase of 57 ± 13% (P = 0.002). In transgenic mice with severe heart failure caused by cardiac-specific overexpression of Gαq, i.p. ITPP increased exercise capacity, with a maximal increase of 63 ± 7% (P = 0.005). Oral administration of ITPP in drinking water increased Hb p50 and maximal exercise capacity (+34 ± 10%; P < 0.002) in normal and failing mice. Consistent with increased tissue oxygen availability, ITPP decreased hypoxia inducible factor-1α mRNA expression in myocardium. It had no effect on myocardial contractility in isolated mouse cardiac myocytes and did not affect arterial blood pressure in vivo in mice. Thus, ITPP decreases the oxygen binding affinity of Hb, increases tissue oxygen delivery, and increases maximal exercise capacity in normal mice and mice with severe heart failure. ITPP is thus an attractive candidate for the therapy of patients with reduced exercise capacity caused by heart failure