Human primary endothelial label-free biochip assay reveals unpredicted functions of plasma serine proteases

Abstract

Tissue-on-a-chip technologies are more and more important in the investigation of cellular function and in the development of novel drugs by allowing the direct screening of substances on human cells. Constituting the inner lining of vessel walls, endothelial cells are the key players in various physiological processes, moreover, they are the first to be exposed to most drugs currently used. However, to date, there is still no appropriate technology for the label-free, real-time and high-throughput monitoring of endothelial function. To this end, we developed an optical biosensor-based endothelial label-free biochip (EnLaB) assay that meets all the above requirements. Using our EnLaB platform, we screened a set of plasma serine proteases as possible endothelial cell activators, and first identified the endothelial cell activating function of three important serine proteases-namely kallikrein, C1r and mannan-binding lectin-associated serine-protease 2 (MASP-2)-and verified these results in well-established functional assays. EnLaB proved to be an effective tool for revealing novel cellular mechanisms as well as for the high-throughput screening of various compounds on endothelial cells.

Advanced Materials and Sensors for Microphysiological Systems: Focus on Electronic and Electrooptical Interfaces

“…The group used the same principle to investigate the role of plasma proteases on endothelial cell layers. [ 147 ] The resonant wavelength shifts in response to the refractive index changes in the evanescent field and are proportional to the optical density above the surface, representing information about the cell–substrate interactions or the presence of biomolecules. [ 148 ]…”

Molecular Dambusters: What Is Behind Hyperpermeability in Bradykinin-Mediated Angioedema?

“…Using a human umbilical vein EC (HUVEC) model, we showed that MASP-2 is also a potent permeability-increasing enzyme. Although both the mechanism and the kinetics are very similar to those of thrombin and MASP-1 [ 98 ], the involvement of PARs in its activity is yet to be proven. We found a significant increase in MASP-2 levels in the 1/3 of patients during angioedema attacks [ 99 ], which supports the notion that MASP-2 may contribute to edema formation.…”

“…Increased leukocyte trafficking and enhanced ICAM-1 and VCAM-1 expression was demonstrated in the presence of PKa [ 101 ], which may modulate permeability indirectly through pro-inflammatory cytokines. PKa can cleave endothelial VE-cadherin in AJs [ 102 ]; moreover, as we have shown, it can activate ECs (possibly via PAR cleavage) and thereby directly increases endothelial permeability [ 98 ].…”

“…C1r is a serine protease indispensable for the complement classical pathway. We showed that C1r can also increase endothelial permeability, but this effect is independent of its enzymatic activity, therefore, it may not be mediated by PARs [ 98 ]. The exact mechanism awaits explanation.…”

Grating-coupled interferometry reveals binding kinetics and affinities of Ni ions to genetically engineered protein layers

“…In the last few decades, label-free optical biosensors have proven their unique capabilities in a wide range of applications from biomolecular interaction analysis (BIA) [1][2][3][4] to whole-cell monitoring [5][6][7][8][9] . Screening the interactions of various analyte-ligand pairs has become essential in drug discovery [10][11][12][13] , assay and sensor development 14,15 as well as for understanding molecular mechanisms in biochemistry and biophysics [16][17][18][19] . Often, the introduction of labels is simply impossible in such measurements, and the application of label-free methods is the only reliable choice.…”