Combating Cardiovascular Disease: Is There a Risk of Foam Cell Formation in Transplanted Angiocompetent Cells Compromising Intended Beneficial Effects of Vascular Regenerative Therapy?.
Authors: Rohde Eva , Aflaki Elma , Froehlich Eleonore , Lanzer Gerhard , Kratky Dagmar , Strunk Dirk 
Published: April 2020

Abstract
Endothelial progenitor cells (EPCs) are the focus of intense clinical research towards vascular regenerative therapy. Somatic vascular EPCs can interact with myelomonocytic and smooth muscle cells modulating the angiogenic response during vascular homeostasis, regeneration and tumour angiogenesis. Interestingly monocytes, either infused freshly after mononuclear cell separation or cultured to induce a pro-angiogenic stimulus prior to re-application, represent the dominant cell fraction in numerous ongoing or already completed clinical trials. Both monocyte-derived macrophages and smooth muscle cells are known to contribute to atherogenesis through lipoprotein endocytosis-mediated foam cell formation. Safety concerns regarding foam cell-related side effects in the course of cellular therapy are evident in atherosclerosis patients. Therefore we examined the foam cell formation potential of typical clinically applied monocyte formulations compared to smooth muscle and endothelial progenitors. Foam cell formation was tested with purified human CD14+ monocytes which were either immediately exposed to 30μg/mL acetylated low-density lipoprotein (acLDL) and cultured for 12 to 72 hours or subjected to a three day pro-angiogenic pre-culture prior to the acLDL exposure. For comparison endothelial colony-forming cells (ECFCs) representing somatic vascular EPCs and multipotent mesenchymal stromal cells (MSCs) as smooth muscle precursors were equally treated in the continuous presence or absence of acLDL. Intracellular lipid accumulation was detected by nile red staining and fluorescence laser scanning microscopy. The number of lipid droplets (LDs) per cell was analyzed using ImageJ software to monitor foam cell formation. Virtual 0.5μm sections were evaluated along the z-axis of a minimum of 40–100 cells per test condition. Depending on cell dimensions, 12 to 30 sections were recorded and LDs were counted in each z-stack. Cellular cholesterol ester and triglyceride content were measured by gas chromatography to further substantiate the pro-atherogenic lipid metabolism. Results revealed intracellular accumulation of bright nile red-fluorescent microvesicles (LDs) in a perinuclear location and size typical for foam cells. Twelve hours of acLDL exposure after a three day pro-angiogenic monocyte pre-culture resulted in a more than three- to 50- fold increased LD count, in comparison to 12h acLDL-exposed fresh monocytes (mean±SD: 42±14; range: 28–58 LDs/cell vs. 5±7; 0–18 LDs/cell; p= 0.027; n=6). Continuous presence of acLDL for 72 hours increased the LD count in cells derived from fresh monocytes two- to 20-fold (20±7; 13–32 LDs/cell; p= 0.046; n=6) but still resulted in decreased numbers of LDs/cell compared to the corresponding pro-angiogenic stimulated monocytes (p=0.046). Surprisingly not only MSCs but also ECFCs displayed foam cell formation potential when subjected to the same permissive conditions in vitro.
These data demonstrate that a pro-angiogenic culture can render human monocytes susceptible to foam cell formation. Furthermore, bone marrow-derived MSCs and vascular EPCs also display foam cell formation potential. We speculate that cellular therapy with “foam cell skewed” monocytes and other modified LDL-susceptible candidate regenerative cells may be ineffective or even of risk to patients with cardiovascular diseases, unless underlying pathologic pro-atherogenic conditions are not reverted appropriately. Our data also strengthen requests to re-examine the role that different cell types play during vascular physio-pathology prior to further clinical trials (as exemplified in Blood109:1801; 2007).