RESUMO
Cardiac cell surface proteins are drug targets and useful biomarkers for discriminating among cellular phenotypes and disease states. Here we developed an analytical platform, CellSurfer, that enables quantitative cell surface proteome (surfaceome) profiling of cells present in limited quantities, and we apply it to isolated primary human heart cells. We report experimental evidence of surface localization and extracellular domains for 1,144 N-glycoproteins, including cell-type-restricted and region-restricted glycoproteins. We identified a surface protein specific for healthy cardiomyocytes, LSMEM2, and validated an anti-LSMEM2 monoclonal antibody for flow cytometry and imaging. Surfaceome comparisons among pluripotent stem cell derivatives and their primary counterparts highlighted important differences with direct implications for drug screening and disease modeling. Finally, 20% of cell surface proteins, including LSMEM2, were differentially abundant between failing and non-failing cardiomyocytes. These results represent a rich resource to advance development of cell type and organ-specific targets for drug delivery, disease modeling, immunophenotyping and in vivo imaging.
RESUMO
Purpose: To investigate the use of tissue plasminogen activator (tPA) and its effects on the ocular proteome as a therapeutic intervention for postoperative inflammation and fibrin formation following intraocular lens (IOL) insertion in a juvenile rabbit model. Methods: Twenty-six rabbits, 6 to 7 weeks old, underwent lensectomy with IOL insertion. Following examination on day 3, 100 µL of either 25 µg of recombinant rabbit tPA or balanced salt solution (control) was injected into the anterior chamber. On postoperative day 4, rabbits underwent examination, and eyes were harvested and fixed for 9.4-Tesla magnetic resonance imaging (MRI). Three masked observers quantified fibrin scar volume using Horos Project software. Aqueous humor (AH) was collected immediately prior to surgery and on postoperative days 3 and 4. Proteins related to coagulation and inflammation were assessed in AH samples using targeted mass spectrometry via parallel reaction monitoring. Results: tPA significantly reduced the volume of fibrin 24 hours following administration compared with control eyes (0.560 mm3 vs. 3.29 mm3; P < 0.0001). Despite the reduced fibrin scar, proteins related to the coagulation and complement cascade were not significantly different following tPA injection. Conclusions: tPA may be a safe candidate for reduction of postoperative fibrin scarring after intraocular surgery. MRI can provide a quantitative value for fibrin volume changes. Translational Relevance: tPA is a candidate to treat ocular fibrin scarring. MRI can quantify the efficacy of treatments in future dose-response studies. Targeted mass spectrometry can provide critical data necessary to help decipher the effect on the abundance of targeted proteins following pharmacological intervention.
