John P. Shapiro

Detection of clinically relevant levels of protein analyte under physiologic buffer using planar field effect transistors

Electrochemical detection of protein binding at physiological salt concentration by planar field effect transistor platforms has yet to be documented convincingly. Here we report detection of streptavidin and clinically relevant levels of biotinylated monokine induced by interferon gamma (MIG) at physiological salt concentrations with AlGaN heterojunction field effect transistors (HFETs). The AlGaN HFETs are functionalized with a silane linker and analyte-specific affinity elements. Polarity of sensor responses is as expected from n-type HFETs to negatively and positively charged analytes. Sensitivity of the HFET sensors increases when salt concentration decreases, and the devices also exhibit dose-dependent responses to analyte. Detection of clinically relevant MIG concentrations at physiological salt levels demonstrates the potential for AlGaN devices to be used in development of in vivo biosensors.

Prevention of rat neonatal cardiomyocyte apoptosis induced by simulated in vitro ischemia and reperfusion

Apoptosis, or programmed cell death, is an active metabolic response to physiological signals or exposure to cytotoxic agents. Recent evidence has shown that the cell death response can be modified by agents presumed to be unrelated to the initial signal, but capable of interfering with the molecular mechanisms of the apoptotic pathway progression. Here we show the results of investigations on the use of a phospholipid-based pharmaceutical preparation for suppression of myocardial damage. First, we show that serum or serum/glucose deprivation, in vitro ischemia with subsequent simulated reperfusion, inhibition of protein synthesis, and treatment with ceramide, staurosporine, adriamycin, cis-platinum and menadione induce apoptotic death in a primary culture of rat neonatal cardiomyocytes. Then we demonstrate that a mixture of specific phospholipids, which has been originally purified from soy flour on the basis of its anti-apoptotic activity, prevents cardiomyocyte death induced by serum or serum/glucose deprivation, by ischemia with subsequent simulated reperfusion, and by ceramide, but not by other cytotoxic treatments. This suggests that ceramide, a lipid secondary messenger which triggers apoptosis induced by some cytotoxic agents, may be involved in the process of signaling ischemia/reperfusion induced apoptotic death of cardiomyocytes. These results further demonstrate that an active pharmaceutical preparation for the suppression of cardiomyocyte death can be formulated based upon a novel strategy of apoptosis modification.

Characterization of glomerular diseases using proteomic analysis of laser capture microdissected glomeruli

The application of molecular techniques to characterize clinical kidney biopsies has the potential to provide insights into glomerular diseases that cannot be revealed by traditional renal pathology. The present work is a proof-of-concept approach to test whether proteomic analysis of glomeruli isolated from clinical biopsies by laser capture microdissection can provide unique information regarding differentially expressed proteins relevant to disease pathogenesis. The proteomes of glomeruli isolated by laser capture microdissection from biopsies of normal kidneys (living-related donor kidneys) were compared with those from patients with diabetic nephropathy, lupus nephritis, and fibronectin glomerulopathy. Glomerular proteins were extracted, trypsin digested, and subjected to liquid chromatography–tandem mass spectrometry for identification and quantitation. Relative to normal glomeruli, all disease-associated glomeruli showed an increased presence of complement components, a marked decline in podocyte-associated proteins, and a decrease in proteins associated with cellular metabolism. Additionally, fibronectin glomerulopathy glomeruli differed from all the other glomeruli because of a significant accumulation of fibronectin and fibulin. This study demonstrates that our method acquires reproducible and quantitative proteomic information from laser capture microdissection isolates that can be used to characterize the molecular features of glomerular diseases.

Long-term biocompatibility of NanoGATE drug delivery implant

The fouling of components and the formation of a fibrotic tissue capsule around subcutaneously implanted medical devices are two major obstacles in developing viable, long-term implantable drug delivery systems. NanoGATE is a subcutaneous implant designed for constant-output passive diffusion of a drug of interest through a silicon nanopore membrane. To this end, we have investigated the long-term in vivo biocompatibility of the NanoGATE implant in terms of the fouling of the nanopore membrane and the formation of a fibrotic tissue capsule around the implant. We have also evaluated how these effects influence diffusion of a lysozyme surrogate from the device once implanted within the vascular compartment of a Sprague-Dawley rat model. Using several model biomolecules such as glucose, lysozyme, and albumin, our studies suggest that silicon nanopore membranes do not foul when implanted subcutaneously for 6 mo. This study also reveals the tissue capsule that naturally forms around the implant does not limit diffusion of molecules with molecular weights on the order of 14.4 kDa at therapeutic delivery rates of tens of micrograms per day. This indicates that our NanoGATE implant should be completely functional in vivo, providing constant release levels of a drug over an extended time period. Thus, by adjusting the release rate to fit the pharmacokinetic clearance profile of the Sprague-Dawley rat, long-term steady-state blood plasma concentrations can be achieved.

Engineering functional protein interfaces for immunologically modified field effect transistor (ImmunoFET) by molecular genetic means

The attachment and interactions of analyte receptor biomolecules at solid–liquid interfaces are critical to development of hybrid biological–synthetic sensor devices across all size regimes. We use protein engineering approaches to engineer the sensing interface of biochemically modified field effect transistor sensors (BioFET). To date, we have deposited analyte receptor proteins on FET sensing channels by direct adsorption, used self-assembled monolayers to tether receptor proteins to planar FET SiO2 sensing gates and demonstrated interface biochemical function and electrical function of the corresponding sensors. We have also used phage display to identify short peptides that recognize thermally grown SiO2. Our interest in these peptides is as affinity domains that can be inserted as translational fusions into receptor proteins (antibody fragments or other molecules) to drive oriented interaction with FET sensing surfaces. We have also identified single-chain fragment variables (scFvs, antibody fragments) that recognize an analyte of interest as potential sensor receptors. In addition, we have developed a protein engineering technology (scanning circular permutagenesis) that allows us to alter protein topography to manipulate the position of functional domains of the protein relative to the BioFET sensing surface.

Nanoparticulate Iron Oxide Contrast Agents for Untargeted and Targeted Cardiovascular Magnetic Resonance Imaging

In cardiology and oncology, early disease detection produces better patient outcomes. Imaging approaches, particularly Mag- netic Resonance Imaging (MRI), are attractive for screening large, mostly disease-free populations because they are noninvasive and pro- duce high-resolution anatomical/morphological images from disease sites. Biochemically targeted nanoparticulate contrast agents (ul- trasmall superparamagnetic iron oxide particles called USPIOs, superparamagnetic iron oxide nanoparticles called SPIONs, cross-linked iron oxides called CLIOs, Gadolinium chelates, and other superparamagnetic or paramagnetic particles) can be administered parenterally to define lesional biochemical/cellular composition, in addition to giving excellent definition of morphological features. Such targeted contrast agents can be considered modular nanobiological devices containing, at minimum, a contrast module and a targeting moiety. Here we review synthesis, surface chemistry and physics of superparamagnetic iron oxide MRI contrast agents (USPIOs and SPIONs) and results to date from the use of biochemically targeted and untargeted iron oxide MRI contrast agents in vivo, in vitro and ex vivo in vascular disease, and to a lesser extent, in oncology applications. We discuss design parameters critical to in vivo use of targeted contrast particles, including parameters influencing particle biodistribution/pharmacokinetics and immunogenicity. We discuss the increasing so- phistication of theranostic contrast agents that provide other physiological services concurrent with their MRI function.

Long-acting progestin-only contraceptives impair endometrial vasculature by inhibiting uterine vascular smooth muscle cell survival

Significance Over a million unintended pregnancies occur in the United States each year because of either discontinuation or misuse of contraceptives. The major reason for discontinuation of long-acting progestin-only contraceptives (LAPCs) is the occurrence of abnormal uterine bleeding (AUB). Uncovering the mechanisms underlying LAPC-induced AUB is essential to prevent their discontinuation. We found that LAPCs reduce proliferation of human and guinea pig endometrial vascular smooth muscle cells (VSMCs), resulting in production of thin-walled hyperdilated fragile microvessels. In cultured VSMCs, chemokine (C-C motif) ligand 2 reverses LAPC-mediated inhibition of VSMC proliferation, suggesting that LAPCs impair endometrial vascular integrity and that chemokine ligand 2 administration may prevent LAPC-induced AUB. Molecular mechanisms responsible for abnormal endometrial vasculature in women receiving long-acting progestin-only contraceptives (LAPCs) are unknown. We hypothesize that LAPCs impair vascular smooth muscle cell (VSMC) and pericyte proliferation and migration producing thin-walled hyperdilated fragile microvessels prone to bleeding. Proliferating cell nuclear antigen (PCNA) and α-smooth muscle actin (αSMA) double-immunostaining assessed VSMC differentiation and proliferation in endometria from women before and after DepoProvera (Depo) treatment and from oophorectomized guinea pigs (OVX-GPs) treated with vehicle, estradiol (E2), medroxyprogesterone acetate (MPA), or E2+MPA. Whole-genome profiling, proliferation, and migration assays were performed on cultured VSMCs treated with MPA or etonogestrel (ETO). Endometrial vessels of Depo-administered women displayed reduced αSMA immunoreactivity and fewer PCNA (+) nuclei among αSMA (+) cells (P < 0.008). Microarray analysis of VSMCs identified several MPA- and ETO-altered transcripts regulated by STAT1 signaling (P < 2.22 × 10−6), including chemokine (C-C motif) ligand 2 (CCL2). Both MPA and ETO reduce VSMC proliferation and migration (P < 0.001). Recombinant CCL2 reversed this progestin-mediated inhibition, whereas a STAT1 inhibitor abolished the CCL2 effect. Similarly, the endometria of MPA treated OVX-GPs displayed decreased αSMA staining and fewer PCNA (+) nuclei in VSMC (P < 0.005). In conclusion, LAPCs promote abnormal endometrial vessel formation by inhibiting VSMC proliferation and migration.

Long-Acting Progestin-Only Contraceptives Enhance Human Endometrial Stromal Cell Expressed Neuronal Pentraxin-1 and Reactive Oxygen Species to Promote Endothelial Cell Apoptosis

CONTEXT Despite the absence of progesterone receptor protein in human endometrial endothelial cells (HEECs), endometria of women receiving long-acting progestin-only contraceptives (LAPCs) display reduced uterine blood flow, elevated reactive oxygen species generation, increased angiogenesis, and irregularly distributed, enlarged, fragile microvessels resulting in abnormal uterine bleeding. OBJECTIVE We propose that paracrine factors from LAPC-treated human endometrial stromal cells (HESCs) impair HEEC functions by shifting the balance between HEEC viability and death in favor of the latter. DESIGN AND SETTING Proliferation, apoptosis, and transcriptome analyses were performed in HEECs treated with conditioned medium supernatant (CMS) derived from HESCs treated with estradiol (E2) ± medroxyprogesterone acetate or etonogestrel under normoxia or hypoxia. Mass spectrometry interrogated the CMS secretome while immunostaining for neuronal pentraxin-1 (NPTX1), cleaved caspase-3, and cytochrome c was performed in cultured HEECs and paired endometria from women using LAPCs. MAIN OUTCOME HEEC apoptosis and its underlying mechanism. RESULTS HESC CMS from E2 + medroxyprogesterone acetate or E2 + etonogestrel incubations under hypoxia induced HEEC apoptosis (P < .05), whereas mass spectrometry of the CMS revealed increased NPTX1 secretion (P < .05). Endothelial cleaved caspase-3 and stromal NPTX1 immunoreactivity were significantly higher in LAPC-treated endometria (P < .001). Transcriptomics revealed AKT signaling inhibition and mitochondrial dysfunction in HEECs incubated with HESC CMS. In vitro analyses proved that CMS decreased HEEC AKT phosphorylation (P < .05) and that recombinant NPTX1 (P < .05) or NPTX1 + H2O2 (P < .001) increase HEEC apoptosis and cytosolic cytochrome c levels. CONCLUSIONS LAPC-enhanced NPTX1 secretion and reactive oxygen species generation in HESCs impair HEEC survival resulting in a loss in vascular integrity, demonstrating a novel paracrine mechanism to explain LAPC-induced abnormal uterine bleeding.

Mass spectrometry identification of potential mediators of progestin-only contraceptive-induced abnormal uterine bleeding in human endometrial stromal cells ☆ ☆☆

OBJECTIVE Thrombin and hypoxia each target human endometrial stromal cells (HESCs) to mediate long-acting progestin-only contraceptive (LAPC)-induced abnormal uterine bleeding (AUB). Thus, the secretome resulting from treatment of primary cultures of HESCs with thrombin or hypoxia was screened by mass spectrometry (MS) to detect potential protein mediators that lead to AUB. STUDY DESIGN Cultured HESCs were primed with estradiol±medroxyprogesterone acetate (MPA) or etonogestrel (ETO), the respective progestins in MPA-injected and ETO-implanted LAPCs, and then treated by incubation with thrombin or under hypoxia. Collected conditioned medium supernatants were used for protein identification and quantitation of potential AUB mediators by liquid chromatography combined with tandem mass spectrometry analysis. Microarray analysis of parallel cultures and immunostaining of endometrial biopsies of LAPC users vs. nonusers corroborated MS results. RESULTS MS identified several proteins displaying changes in expression levels from either thrombin or hypoxia treatments that are integral to angiogenesis or extracellular matrix formation. Several MS-identified proteins were confirmed by mRNA microarray analysis. Overexpressed stanniocalcin-1 (STC-1) was observed in endometrium of LAPC users. Unlike controls, all LAPC users displayed endometrial tubal metaplasia (ETM). CONCLUSIONS MS analysis identified many proteins that can affect angiogenesis or vessel integrity, thereby contributing to AUB. Confirmation of STC-1 overexpression in LAPC users and microarray data supports the validity of the MS data and suggests STC-1 involvement in AUB. The discovery of ETM in LAPC users indicates that LAPC-related side effects extend beyond AUB. The results presented here demonstrate a complex biological response to LAPC use. IMPLICATIONS MS identified several HESC secreted proteins deregulated by thrombin and hypoxia that may mediate LAPC-induced AUB. The revelation of overexpressed STC-1 by combined in vivo and in vitro observations identifies a potential target for future studies to prevent or minimize LAPC-induced AUB.

Thrombin impairs human endometrial endothelial angiogenesis; implications for progestin-only contraceptive-induced abnormal uterine bleeding.

OBJECTIVE Progestin-only contraceptives induce abnormal uterine bleeding, accompanied by prothrombin leakage from dilated endometrial microvessels and increased thrombin generation by human endometrial stromal cell (HESC)-expressed tissue factor. Initial studies of the thrombin-treated HESC secretome identified elevated levels of cleaved chondroitin sulfate proteoglycan 4 (CSPG4), impairing pericyte-endothelial interactions. Thus, we investigated direct and CSPG4-mediated effects of thrombin in eliciting abnormal uterine bleeding by disrupting endometrial angiogenesis. STUDY DESIGN Liquid chromatography/tandem mass spectrometry, enzyme-linked immunosorbent assay (ELISA) and quantitative real-time-polymerase chain reaction (PCR) evaluated conditioned medium supernatant and cell lysates from control versus thrombin-treated HESCs. Pre- and post-Depo medroxyprogesterone acetate (DMPA)-administered endometria were immunostained for CSPG4. Proliferation, apoptosis and tube formation were assessed in human endometrial endothelial cells (HEECs) incubated with recombinant human (rh)-CSPG4 or thrombin or both. RESULTS Thrombin induced CSPG4 protein expression in cultured HESCs as detected by mass spectrometry and ELISA (p<.02, n=3). Compared to pre-DMPA endometria (n=5), stromal cells in post-DMPA endometria (n=5) displayed stronger CSPG4 immunostaining. In HEEC cultures (n=3), total tube-formed mesh area was significantly higher in rh-CSPG4 versus control (p<.05). However, thrombin disrupted HEEC tube formation by a concentration- and time-dependent reduction of angiogenic parameters (p<.05), whereas CSPG4 co-treatment did not reverse these thrombin-mediated effects. CONCLUSION These results suggest that disruption of HEEC tube formation by thrombin induces aberrant angiogenesis and abnormal uterine bleeding in DMPA users. IMPLICATIONS Mass spectrometry analysis identified several HESC-secreted proteins regulated by thrombin. Therapeutic agents blocking angiogenic effects of thrombin in HESCs can prevent or minimize progestin-only contraceptive-induced abnormal uterine bleeding.