Challenges in Detection of Serum Oncoprotein: Relevance to Breast Cancer Diagnostics.

Breast cancer is a highly prevalent malignancy that shows improved outcomes with earlier diagnosis. Current screening and monitoring methods have improved survival rates, but the limitations of these approaches have led to the investigation of biomarker evaluation to improve early diagnosis and treatment monitoring. The enzyme-linked immunosorbent assay (ELISA) is a specific and robust technique ideally suited for the quantification of protein biomarkers from blood or its constituents. The continued clinical relevancy of this assay format will require overcoming specific technical challenges, including the ultra-sensitive detection of trace biomarkers and the circumventing of potential assay interference due to the expanding use of monoclonal antibody (mAb) therapeutics. Approaches to increasing the sensitivity of ELISA have been numerous and include employing more sensitive substrates, combining ELISA with the polymerase chain reaction (PCR), and incorporating nanoparticles as shuttles for detection antibodies and enzymes. These modifications have resulted in substantial boosts in the ability to detect extremely low levels of protein biomarkers, with some systems reliably detecting antigen at sub-femtomolar concentrations. Extensive utilization of mAb therapies in oncology has presented an additional contemporary challenge for ELISA, particularly when both therapeutic and assay antibodies target the same protein antigen. Resolution of issues such as epitope overlap and steric hindrance requires a rational approach to the design of diagnostic antibodies that takes advantage of modern antibody generation pipelines, epitope binning techniques and computational methods to strategically target biomarker epitopes. This review discusses technical strategies in ELISA implemented to date and their feasibility to address current constraints on sensitivity and problems with interference in the clinical setting. The impact of these recent advancements will depend upon their transformation from research laboratory protocols into facile, reliable detection systems that can ideally be replicated in point-of-care devices to maximize utilization and transform both the diagnostic and therapeutic monitoring landscape.

Diminished ketone interconversion, hepatic TCA cycle flux, and glucose production in D-ß-hydroxybutyrate dehydrogenase hepatocyte-deficient mice.

OBJECTIVE: Throughout the last decade, interest has intensified in intermittent fasting, ketogenic diets, and exogenous ketone therapies as prospective health-promoting, therapeutic, and performance-enhancing agents. However, the regulatory roles of ketogenesis and ketone metabolism on liver homeostasis remain unclear. Therefore, we sought to develop a better understanding of the metabolic consequences of hepatic ketone body metabolism by focusing on the redox-dependent interconversion of acetoacetate (AcAc) and D-ß-hydroxybutyrate (D-ßOHB). METHODS: Using targeted and isotope tracing high-resolution liquid chromatography-mass spectrometry, dual stable isotope tracer nuclear magnetic resonance spectroscopy-based metabolic flux modeling, and complementary physiological approaches in novel cell type-specific knockout mice, we quantified the roles of hepatocyte D-ß-hydroxybutyrate dehydrogenase (BDH1), a mitochondrial enzyme required for NAD+/NADH-dependent oxidation/reduction of ketone bodies. RESULTS: Exogenously administered AcAc is reduced to D-ßOHB, which increases hepatic NAD+/NADH ratio and reflects hepatic BDH1 activity. Livers of hepatocyte-specific BDH1-deficient mice did not produce D-ßOHB, but owing to extrahepatic BDH1, these mice nonetheless remained capable of AcAc/D-ßOHB interconversion. Compared to littermate controls, hepatocyte-specific BDH1 deficient mice exhibited diminished liver tricarboxylic acid (TCA) cycle flux and impaired gluconeogenesis, but normal hepatic energy charge overall. Glycemic recovery after acute insulin challenge was impaired in knockout mice, but they were not more susceptible to starvation-induced hypoglycemia. CONCLUSIONS: Ketone bodies influence liver homeostasis. While liver BDH1 is not required for whole body equilibration of AcAc and D-ßOHB, loss of the ability to interconvert these ketone bodies in hepatocytes results in impaired TCA cycle flux and glucose production. Therefore, through oxidation/reduction of ketone bodies, BDH1 is a significant contributor to hepatic mitochondrial redox, liver physiology, and organism-wide ketone body homeostasis.

Hepatocyte-Macrophage Acetoacetate Shuttle Protects against Tissue Fibrosis.

Metabolic plasticity has been linked to polarized macrophage function, but mechanisms connecting specific fuels to tissue macrophage function remain unresolved. Here we apply a stable isotope tracing, mass spectrometry-based untargeted metabolomics approach to reveal the metabolome penetrated by hepatocyte-derived glucose and ketone bodies. In both classically and alternatively polarized macrophages, [13C]acetoacetate (AcAc) labeled ∼200 chemical features, but its reduced form D-[13C]ß-hydroxybutyrate (D-ßOHB) labeled almost none. [13C]glucose labeled ∼500 features, and while unlabeled AcAc competed with only ∼15% of them, the vast majority required the mitochondrial enzyme succinyl-coenzyme A-oxoacid transferase (SCOT). AcAc carbon labeled metabolites within the cytoplasmic glycosaminoglycan pathway, which regulates tissue fibrogenesis. Accordingly, livers of mice lacking SCOT in macrophages were predisposed to accelerated fibrogenesis. Exogenous AcAc, but not D-ßOHB, ameliorated diet-induced hepatic fibrosis. These data support a hepatocyte-macrophage ketone shuttle that segregates AcAc from D-ßOHB, coordinating the fibrogenic response to hepatic injury via mitochondrial metabolism in tissue macrophages.

Endothelial Wnt/ß-catenin signaling reduces immune cell infiltration in multiple sclerosis.

Disruption of the blood-brain barrier (BBB) is a defining and early feature of multiple sclerosis (MS) that directly damages the central nervous system (CNS), promotes immune cell infiltration, and influences clinical outcomes. There is an urgent need for new therapies to protect and restore BBB function, either by strengthening endothelial tight junctions or suppressing endothelial vesicular transcytosis. Although wingless integrated MMTV (Wnt)/ß-catenin signaling plays an essential role in BBB formation and maintenance in healthy CNS, its role in BBB repair in neurologic diseases such as MS remains unclear. Using a Wnt/ß-catenin reporter mouse and several downstream targets, we demonstrate that the Wnt/ß-catenin pathway is up-regulated in CNS endothelial cells in both human MS and the mouse model experimental autoimmune encephalomyelitis (EAE). Increased Wnt/ß-catenin activity in CNS blood vessels during EAE progression correlates with up-regulation of neuronal Wnt3 expression, as well as breakdown of endothelial cell junctions. Genetic inhibition of the Wnt/ß-catenin pathway in CNS endothelium before disease onset exacerbates the clinical presentation of EAE, CD4+ T-cell infiltration into the CNS, and demyelination by increasing expression of vascular cell adhesion molecule-1 and the transcytosis protein Caveolin-1 and promoting endothelial transcytosis. However, Wnt signaling attenuation does not affect the progressive degradation of tight junction proteins or paracellular BBB leakage. These results suggest that reactivation of Wnt/ß-catenin signaling in CNS vessels during EAE/MS partially restores functional BBB integrity and limits immune cell infiltration into the CNS.

The role of angiogenesis in the pathology of multiple sclerosis.

Angiogenesis, or the growth of new blood vessels from existing vasculature, is critical for the proper development of many organs. This process is inhibited and tightly regulated in adults, once endothelial cells have acquired organ-specific properties. Within the central nervous system (CNS), angiogenesis and acquisition of blood-brain barrier (BBB) properties by endothelial cells is essential for CNS function. However, the role of angiogenesis in CNS pathologies associated with impaired barrier function remains unclear. Although vessel abnormalities characterized by abnormal barrier function are well documented in multiple sclerosis (MS), a demyelinating disease of the CNS resulting from an immune cell attack on oligodendrocytes, histological analysis of human MS samples has shown that angiogenesis is prevalent in and around the demyelinating plaques. Experiments using an animal model that mimics several features of human MS, Experimental Autoimmune Encephalomyelitis (EAE), have confirmed these human pathological findings and shed new light on the contribution of pre-symptomatic angiogenesis to disease progression. The CNS-infiltrating inflammatory cells that are a hallmark of both MS and EAE secrete several factors that not only contribute to exacerbating the inflammatory process but also promote and stimulate angiogenesis. Moreover, chemical or biological inhibitors that directly or indirectly block angiogenesis provide clinical benefits for disease progression. While the precise mechanism of action for these inhibitors is unknown, preventing pathological angiogenesis during EAE progression holds great promise for developing effective treatment strategies for human MS.

Elevated protein kinase C-δ contributes to aneurysm pathogenesis through stimulation of apoptosis and inflammatory signaling.

OBJECTIVE: Apoptosis of smooth muscle cells (SMCs) is a prominent pathological characteristic of abdominal aortic aneurysm (AAA). We have previously shown that SMC apoptosis stimulates proinflammatory signaling in a mouse model of AAA. Here, we test whether protein kinase C-δ (PKCδ), an apoptotic mediator, participates in the pathogenesis of AAA by regulating apoptosis and proinflammatory signals. METHODS AND RESULTS: Mouse experimental AAA is induced by perivascular administration of CaCl(2). Mice deficient in PKCδ exhibit a profound reduction in aneurysmal expansion, SMC apoptosis, and transmural inflammation as compared with wild-type littermates. Delivery of PKCδ to the aortic wall of PKCδ(-/-) mice restores aneurysm, whereas overexpression of a dominant negative PKCδ mutant in the aorta of wild-type mice attenuates aneurysm. In vitro, PKCδ(-/-) aortic SMCs exhibit significantly impaired monocyte chemoattractant protein-1 production. Ectopic administration of recombinant monocyte chemoattractant protein-1 to the arterial wall of PKCδ(-/-) mice restores inflammatory response and aneurysm development. CONCLUSIONS: PKCδ is an important signaling mediator for SMC apoptosis and inflammation in a mouse model of AAA. By stimulating monocyte chemoattractant protein-1 expression in aortic SMCs, upregulated PKCδ exacerbates the inflammatory process, in turn perpetuating elastin degradation and aneurysmal dilatation. Inhibition of PKCδ may serve as a potential therapeutic strategy for AAA.

Accelerated aneurysmal dilation associated with apoptosis and inflammation in a newly developed calcium phosphate rodent abdominal aortic aneurysm model.

OBJECTIVE: The calcium chloride (CaCl(2)) model is a widely accepted rodent model for abdominal aortic aneurysms (AAAs). Calcium deposition, mainly consisting of calcium phosphate (CaPO(4)) crystals, has been reported to exist in human and experimental aneurysms. CaPO(4) crystals have been used for in vitro DNA transfection by mixing CaCl(2) and phosphate-buffered saline (PBS). Here, we describe accelerated aneurysm formation resulting from a modification of the CaCl(2) model. METHODS: A modified CaCl(2) model, the CaPO(4) model, was created by applying PBS onto the mouse infrarenal aorta after CaCl(2) treatment. Morphologic, histologic, and immunohistochemical analyses were performed on arteries treated with the CaPO(4) model and the conventional CaCl(2) model as the control. In vitro methods were performed using a mixture of CaCl(2) and PBS to create CaPO(4) crystals. CaPO(4)- induced apoptosis of primary cultured mouse vascular smooth muscle cells (VSMCs) was measured by DNA fragmentation enzyme-linked immunosorbent assay. RESULTS: The CaPO(4) model produces AAA, defined as an increase of ≥50% in the diameter of the aorta, faster than in the CaCl(2) model. The CaPO(4) model showed significantly larger aneurysmal dilation at 7, 28, and 42 days, as reflected by a maximum diameter (measured in mm) fold-change of 1.69 ± 0.07, 1.99 ± 0.14, and 2.13 ± 0.09 vs 1.22 ± 0.04, 1.48 ± 0.07, and 1.68 ± 0.06 in a CaCl(2) model, respectively (n = 6; P < .05). A semiquantitative grading analysis of elastin fiber integrity at 7 days revealed a significant increase in elastin degradation in the CaPO(4) model compared with the CaCl(2) model (2.7 ± 0.2 vs 1.5 ± 0.2; n = 6; P < .05). A significantly higher level of apoptosis occurred in the CaPO(4) model (apoptosis index at 1, 2, and 3 days postsurgery: 0.26 ± 0.14, 0.37 ± 0.14, and 0.33 ± 0.08 vs 0.012 ± 0.10, 0.15 ± 0.02, and 0.12 ± 0.05 in the conventional CaCl(2) model; n = 3; P < .05). An enhancement of macrophage infiltration and calcification was also observed at 3 and 7 days in the CaPO(4) model. CaPO(4) induced approximately 3.7 times more apoptosis in VSMCs than a mixture of CaCl(2) (n = 4; P < .0001) in vitro. CONCLUSIONS: The CaPO(4) model accelerates aneurysm formation with the enhancement of apoptosis, macrophage infiltration, and calcium deposition. This modified model, with its rapid and robust dilation, can be used as a new model for AAAs.

Protein kinase C δ regulates the release of collagen type I from vascular smooth muscle cells via regulation of Cdc42.

Collagen type I is the most abundant component of extracellular matrix in the arterial wall. Mice knocked out for the protein kinase C δ gene (PKCδ KO) show a marked reduction of collagen I in the arterial wall. The lack of PKCδ diminished the ability of arterial smooth muscle cells (SMCs) to secrete collagen I without significantly altering the intracellular collagen content. Moreover, the unsecreted collagen I molecules accumulate in large perinuclear puncta. These perinuclear structures colocalize with the trans-Golgi network (TGN) marker TGN38 and to a lesser degree with cis-Golgi marker (GM130) but not with early endosomal marker (EEA1). Associated with diminished collagen I secretion, PKCδ KO SMCs exhibit a significant reduction in levels of cell division cycle 42 (Cdc42) protein and mRNA. Restoring PKCδ expression partially rescues Cdc42 expression and collagen I secretion in PKCδ KO SMCs. Inhibition of Cdc42 expression or activity with small interfering RNA or secramine A in PKCδ WT SMCs eliminates collagen I secretion. Conversely, restoring Cdc42 expression in PKCδ KO SMCs enables collagen I secretion. Taken together, our data demonstrate that PKCδ mediates collagen I secretion from SMCs, likely through a Cdc42-dependent mechanism.

The novel function of advanced glycation end products in regulation of MMP-9 production.

BACKGROUND: Advanced glycation end products (AGEs), formed from proteins and peptides by nonenzymatic glycoxidation after contact with aldose sugars, have been implicated in the pathogenesis of age-related cardiac and vascular dysfunction. Our previous study demonstrated significantly elevated levels of AGE and the receptor for AGE (RAGE) in human abdominal aortic aneurysm (AAA) tissues. Inhibition of AGE signaling by targeted gene deletion of RAGE markedly reduced the development of aneurysm in a mouse model of AAA. We also showed that AGE may stimulate aneurysm formation by promoting metalloproteinase (MMP)-9 expression. In this study, we investigated the molecular mechanism underlying this novel function of AGE. METHODS: The murine macrophage cell line RAW 264.7 was pretreated with AGE, TGF-ß, and MAPK inhibitors. The protein was collected for Western blot analysis. Culture supernatants were collected to determine MMP-9 activity by gelatin zymography. RESULTS: We found that AGE induced the production of MMP-9 in macrophages in a dose-dependent manner. This induction of MMP-9 was markedly diminished by pretreatment with TGF-ß. To delineate the underlying molecular mechanism, we showed that AGE increased phosphorylation of p44/42 ERK, p38, JNK, and PI3K in macrophages. Moreover, AGE induced active p65 subunit of NF- κB. Inhibition of ERK (UO126) or p38 (SB203580), but not PI3K (LY294002 or wortmannin), blocked AGE-induced MMP-9 expression. In contrast, inhibition of JNK (SP-600125) significantly enhanced the stimulatory effect of AGE on MMP-9. Furthermore, TGF-ß suppressed AGE-induced expression of the active p65 subunit of NF-κB. CONCLUSIONS: Our data indicate that AGE induces MMP-9 through activation of ERK, p38 mitogen-activated protein and NF-κB, a pathway that is antagonized by TGF-ß. This finding in conjunction with previously reported AGE functions in inflammation suggests that anti-AGE therapies could be effective in the prevention of human AAA development and progression.

Effects of caspase inhibitor on angiotensin II-induced abdominal aortic aneurysm in apolipoprotein E-deficient mice.

OBJECTIVE: The presence of apoptotic markers is a prominent histological feature of abdominal aortic aneurysm. To understand the role of apoptosis in the pathogenesis of this common vascular disease, we tested the effect of the pan-caspase inhibitor quinoline-Val-Asp-difluorophenoxymethylketone (Q-VD-OPh) on aneurysm formation using a mouse angiotensin II (Ang II) model. METHODS AND RESULTS: Ang II in apolipoprotein E-deficient mice significantly induced medial cell apoptosis 3 days after infusion at the aortic region, eventually becoming aneurismal. A daily administration of 20 mg/kg per day Q-VD-OPh starting 6 hours before Ang II infusion reduced aneurysm incidence from 83.3% to 16.7% and maximal aortic diameter from 2.43+/-0.29 mm to 1.58+/-0.18 mm. The caspase inhibitor treated mice showed profoundly diminished levels of medial apoptosis and inflammation. In contrast, administration of Q-VD-OPh starting 7 days after Ang II infusion had no significant impact on aneurysm development. In vitro, media conditioned by Ang II-treated smooth muscle cells (SMCs) stimulated macrophage chemotaxis in a caspase-dependent manner. Inhibition of monocyte chemoattractant protein-1 (MCP-1) in the conditioned media via a neutralizing antibody completely blocked the ability of conditioned media to attract macrophages. CONCLUSIONS: These results indicate that medial SMC apoptosis may contribute to vascular inflammation and thus aneurysm formation, in part through production of MCP-1.