Ryan M. Burke

Two-Photon and Second Harmonic Microscopy in Clinical and Translational Cancer Research

Application of two-photon microscopy (TPM) to translational and clinical cancer research has burgeoned over the last several years, as several avenues of pre-clinical research have come to fruition. In this review, we focus on two forms of TPM—two-photon excitation fluorescence microscopy, and second harmonic generation microscopy—as they have been used for investigating cancer pathology in ex vivo and in vivo human tissue. We begin with discussion of two-photon theory and instrumentation particularly as applicable to cancer research, followed by an overview of some of the relevant cancer research literature in areas that include two-photon imaging of human tissue biopsies, human skin in vivo, and the rapidly developing technology of two-photon microendoscopy. We believe these and other evolving two-photon methodologies will continue to help translate cancer research from the bench to the bedside, and ultimately bring minimally invasive methods for cancer diagnosis and treatment to therapeutic reality.

Second harmonic properties of tumor collagen: determining the structural relationship between reactive stroma and healthy stroma.

We utilize the polarization and directionality of light emitted by fibrillar collagen via second harmonic generation to determine structural relationships between collagen in mouse mammary tumor models and the healthy mammary fat pad. In spite of the aberrations in collagen production and degradation that are the hallmarks of tumor stroma, we find that the characteristic angle of SHG scatterers within collagen fibrils, and the spatial extent over which they are appropriately ordered for SHG production, are the same in tumor and healthy collagen. This suggests that the SHG-producing subpopulation of collagen is unaffected by the altered collagen synthesis of the tumor stroma, and protected from its aberrant degradative environment.

Thioredoxin-Interacting Protein Is a Biomechanical Regulator of Src Activity Key Role in Endothelial Cell Stress Fiber Formation

Rationale: Fluid shear stress differentially regulates endothelial cell stress fiber formation with decreased stress fibers in areas of disturbed flow compared with steady flow areas. Importantly, stress fibers are critical for several endothelial cell functions including cell shape, mechano-signal transduction, and endothelial cell–cell junction integrity. A key mediator of steady flow–induced stress fiber formation is Src that regulates downstream signaling mediators such as phosphorylation of cortactin, activity of focal adhesion kinase, and small GTPases. Objective: Previously, we showed that thioredoxin-interacting protein (TXNIP, also VDUP1 [vitamin D upregulated protein 1] and TBP-2 [thioredoxin binding protein 2]) was regulated by fluid shear stress; TXNIP expression was increased in disturbed flow compared with steady flow areas. Although TXNIP was originally characterized for its role in redox and metabolic cellular functions, recent reports show important scaffold functions related to its α-arrestin structure. Based on these findings, we hypothesized that TXNIP acts as a biomechanical sensor that regulates Src kinase activity and stress fiber formation. Methods and Results: Using en face immunohistochemistry of the aorta and cultured endothelial cells, we show inverse relationship between TXNIP expression and Src activity. Specifically, steady flow increased Src activity and stress fiber formation, whereas it decreased TXNIP expression. In contrast, disturbed flow had opposite effects. We studied the role of TXNIP in regulating Src homology phosphatase-2 plasma membrane localization and vascular endothelial cadherin binding because Src homology phosphatase-2 indirectly regulates dephosphorylation of Src tyrosine 527 that inhibits Src activity. Using immunohistochemistry and immunoprecipitation, we found that TXNIP prevented Src homology phosphatase-2–vascular endothelial cadherin interaction. Conclusions: In summary, these data characterize a fluid shear stress–mediated mechanism for stress fiber formation that involves a TXNIP-dependent vascular endothelial cadherin–Src homology phosphatase-2–Src pathway. # Novelty and Significance {#article-title-59}Rationale: Fluid shear stress differentially regulates endothelial cell stress fiber formation with decreased stress fibers in areas of disturbed flow compared with steady flow areas. Importantly, stress fibers are critical for several endothelial cell functions including cell shape, mechano-signal transduction, and endothelial cell–cell junction integrity. A key mediator of steady flow–induced stress fiber formation is Src that regulates downstream signaling mediators such as phosphorylation of cortactin, activity of focal adhesion kinase, and small GTPases. Objective: Previously, we showed that thioredoxin-interacting protein (TXNIP, also VDUP1 [vitamin D upregulated protein 1] and TBP-2 [thioredoxin binding protein 2]) was regulated by fluid shear stress; TXNIP expression was increased in disturbed flow compared with steady flow areas. Although TXNIP was originally characterized for its role in redox and metabolic cellular functions, recent reports show important scaffold functions related to its &agr;-arrestin structure. Based on these findings, we hypothesized that TXNIP acts as a biomechanical sensor that regulates Src kinase activity and stress fiber formation. Methods and Results: Using en face immunohistochemistry of the aorta and cultured endothelial cells, we show inverse relationship between TXNIP expression and Src activity. Specifically, steady flow increased Src activity and stress fiber formation, whereas it decreased TXNIP expression. In contrast, disturbed flow had opposite effects. We studied the role of TXNIP in regulating Src homology phosphatase-2 plasma membrane localization and vascular endothelial cadherin binding because Src homology phosphatase-2 indirectly regulates dephosphorylation of Src tyrosine 527 that inhibits Src activity. Using immunohistochemistry and immunoprecipitation, we found that TXNIP prevented Src homology phosphatase-2–vascular endothelial cadherin interaction. Conclusions: In summary, these data characterize a fluid shear stress–mediated mechanism for stress fiber formation that involves a TXNIP-dependent vascular endothelial cadherin–Src homology phosphatase-2–Src pathway.

Tumor-associated macrophages and stromal TNF-α regulate collagen structure in a breast tumor model as visualized by second harmonic generation

Abstract. Collagen fibers can be imaged with second harmonic generation (SHG) and are associated with efficient tumor cell locomotion. Preferential locomotion along these fibers correlates with a more aggressively metastatic phenotype, and changes in SHG emission properties accompany changes in metastatic outcome. We therefore attempted to elucidate the cellular and molecular machinery that influences SHG in order to understand how the microstructure of tumor collagen fibers is regulated. By quantifying SHG and immunofluorescence (IF) from tumors grown in mice with and without stromal tumor necrosis factor (TNF)-α and in the presence or absence of tumor-associated macrophages (TAMs), we determined that depletion of TAMs alters tumor collagen fibrillar microstructure as quantified by SHG and IF. Furthermore, we determined that abrogation of TNF-α expression by tumor stromal cells also alters fibrillar microstructure and that subsequent depletion of TAMs has no further effect. In each case, metastatic burden correlated with optical readouts of collagen microstructure. Our results implicate TAMs and stromal TNF-α as regulators of breast tumor collagen microstructure and suggest that this regulation plays a role in tumor metastasis. Furthermore, these results indicate that quantification of SHG represents a useful strategy for evaluating the cells and molecular pathways responsible for manipulating fibrillar collagen in breast tumor models.

Intima modifier locus 2 controls endothelial cell activation and vascular permeability

Carotid intima formation is a significant risk factor for cardiovascular disease. C3H/FeJ (C3H/F) and SJL/J (SJL) inbred mouse strains differ in susceptibility to immune and vascular traits. Using a congenic approach we demonstrated that the Intima modifier 2 (Im2) locus on chromosome 11 regulates leukocyte infiltration. We sought to determine whether inflammation was due to changes in circulating immune cells or activation of vascular wall cells in genetically pure Im2 (C3H/F.SJL.11.1) mice. Complete blood counts showed no differences in circulating monocytes between C3H/F and C3H/F.SJL.11.1 compared with SJL mice. Aortic vascular cell adhesion molecule-1 (VCAM-1) total protein levels were dramatically increased in SJL and C3H/F.SJL.11.1 compared with C3H/F mice. Immunostaining of aortic endothelial cells (EC) showed a significant increase in VCAM-1 expression in SJL and C3H/F.SJL.11.1 compared with C3H/F under steady flow conditions. Immunostaining of EC membranes revealed a significant decrease in EC size in SJL and C3H/F.SJL.11.1 vs. C3H/F in regions of disturbed flow. Vascular permeability was significantly higher in C3H/F.SJL.11.1 compared with C3H/F. Our results indicate that Im2 regulation of leukocyte infiltration is mediated by EC inflammation and permeability. RNA sequencing and pathway analyses comparing genes in the Im2 locus to C3H/F provide insight into candidate genes that regulate vascular wall inflammation and permeability highlighting important genetic mechanisms that control vascular intima in response to injury.

In vivo imaging of tumors Using multiphoton fluorescence and second harmonic generation

In this tutorial we will discuss how epifluorescence microscopy, confocal laser-scanning microscopy, and multiphoton laser-scanning microscopy are used to provide a detailed understanding of the pathophysiology of living tumors.

Small proline-rich protein 2B drives stress-dependent p53 degradation and fibroblast proliferation in heart failure

Significance Heart disease is associated with the development of fibrosis, a type of scarring that impedes cardiac function. The primary cellular source of cardiac fibrosis is the resident cardiac fibroblast. We found that cardiac fibroblasts from human heart failure patients or a mouse model of heart disease express excessive amounts of the SPRR2B protein. We provide evidence that SPRR2B is a signal-responsive regulatory subunit of the p53 ubiquitination complex that stimulates the destruction of p53 and the accumulation of pathological fibroblasts. This study defines a unique mechanism of cell cycle control that is dysfunctional in heart disease and may drive the development pathological fibrosis. Heart disease is associated with the accumulation of resident cardiac fibroblasts (CFs) that secrete extracellular matrix (ECM), leading to the development of pathological fibrosis and heart failure. However, the mechanisms underlying resident CF proliferation remain poorly defined. Here, we report that small proline-rich protein 2b (Sprr2b) is among the most up-regulated genes in CFs during heart disease. We demonstrate that SPRR2B is a regulatory subunit of the USP7/MDM2-containing ubiquitination complex. SPRR2B stimulates the accumulation of MDM2 and the degradation of p53, thus facilitating the proliferation of pathological CFs. Furthermore, SPRR2B phosphorylation by nonreceptor tyrosine kinases in response to TGF-β1 signaling and free-radical production potentiates SPRR2B activity and cell cycle progression. Knockdown of the Sprr2b gene or inhibition of SPRR2B phosphorylation attenuates USP7/MDM2 binding and p53 degradation, leading to CF cell cycle arrest. Importantly, SPRR2B expression is elevated in cardiac tissue from human heart failure patients and correlates with the proliferative state of patient-derived CFs in a process that is reversed by insulin growth factor-1 signaling. These data establish SPRR2B as a unique component of the USP7/MDM2 ubiquitination complex that drives p53 degradation, CF accumulation, and the development of pathological cardiac fibrosis.

β-Adrenergic Blockade in Ischemia-Reperfusion Injury: βARKing Up a New Tree∗

SEE PAGE 958 C oronary artery occlusion leads to myocardial infarction (MI) and subsequent cell death in ischemic cardiac tissue. Recent clinical advances in the treatment of MI, including rapid reperfusion and aggressive use of antiplatelet agents, limit the extent of cell death and save countless patients who would have died just a few decades ago (1). However, even after successful reperfusion, tissue lost during an MI is never repopulated with functioning cardiomyocytes. Instead, fibroblasts adjacent to the ischemic region proliferate, infiltrate the infarct, and become activated. Activated fibroblasts, also called myofibroblasts, secrete extracellular matrix (ECM) that partially restores structural integrity and prevents cardiac rupture. Although scar formation is initially a beneficial response to cardiac insult and is essential for cardiac repair, unrestrained fibroblast activity leads to expansion of fibrosis, culminating in heart failure (HF). Fueled by epidemics of obesity and diabetes that stimulate atherosclerotic narrowing of coronary vessels and ischemic heart disease, the incidence of HF has skyrocketed and is projected to affect 1 in 33 U.S. citizens by 2030 (2). The negative effect of cardiac scarring on heart function is multifactorial (3). Interstitial fibrosis is

Thioredoxin-Interacting Protein Is a Biomechanical Regulator of Src ActivityNovelty and Significance: Key Role in Endothelial Cell Stress Fiber Formation

Rationale: Fluid shear stress differentially regulates endothelial cell stress fiber formation with decreased stress fibers in areas of disturbed flow compared with steady flow areas. Importantly, stress fibers are critical for several endothelial cell functions including cell shape, mechano-signal transduction, and endothelial cell–cell junction integrity. A key mediator of steady flow–induced stress fiber formation is Src that regulates downstream signaling mediators such as phosphorylation of cortactin, activity of focal adhesion kinase, and small GTPases. Objective: Previously, we showed that thioredoxin-interacting protein (TXNIP, also VDUP1 [vitamin D upregulated protein 1] and TBP-2 [thioredoxin binding protein 2]) was regulated by fluid shear stress; TXNIP expression was increased in disturbed flow compared with steady flow areas. Although TXNIP was originally characterized for its role in redox and metabolic cellular functions, recent reports show important scaffold functions related to its α-arrestin structure. Based on these findings, we hypothesized that TXNIP acts as a biomechanical sensor that regulates Src kinase activity and stress fiber formation. Methods and Results: Using en face immunohistochemistry of the aorta and cultured endothelial cells, we show inverse relationship between TXNIP expression and Src activity. Specifically, steady flow increased Src activity and stress fiber formation, whereas it decreased TXNIP expression. In contrast, disturbed flow had opposite effects. We studied the role of TXNIP in regulating Src homology phosphatase-2 plasma membrane localization and vascular endothelial cadherin binding because Src homology phosphatase-2 indirectly regulates dephosphorylation of Src tyrosine 527 that inhibits Src activity. Using immunohistochemistry and immunoprecipitation, we found that TXNIP prevented Src homology phosphatase-2–vascular endothelial cadherin interaction. Conclusions: In summary, these data characterize a fluid shear stress–mediated mechanism for stress fiber formation that involves a TXNIP-dependent vascular endothelial cadherin–Src homology phosphatase-2–Src pathway. # Novelty and Significance {#article-title-59}Rationale: Fluid shear stress differentially regulates endothelial cell stress fiber formation with decreased stress fibers in areas of disturbed flow compared with steady flow areas. Importantly, stress fibers are critical for several endothelial cell functions including cell shape, mechano-signal transduction, and endothelial cell–cell junction integrity. A key mediator of steady flow–induced stress fiber formation is Src that regulates downstream signaling mediators such as phosphorylation of cortactin, activity of focal adhesion kinase, and small GTPases. Objective: Previously, we showed that thioredoxin-interacting protein (TXNIP, also VDUP1 [vitamin D upregulated protein 1] and TBP-2 [thioredoxin binding protein 2]) was regulated by fluid shear stress; TXNIP expression was increased in disturbed flow compared with steady flow areas. Although TXNIP was originally characterized for its role in redox and metabolic cellular functions, recent reports show important scaffold functions related to its &agr;-arrestin structure. Based on these findings, we hypothesized that TXNIP acts as a biomechanical sensor that regulates Src kinase activity and stress fiber formation. Methods and Results: Using en face immunohistochemistry of the aorta and cultured endothelial cells, we show inverse relationship between TXNIP expression and Src activity. Specifically, steady flow increased Src activity and stress fiber formation, whereas it decreased TXNIP expression. In contrast, disturbed flow had opposite effects. We studied the role of TXNIP in regulating Src homology phosphatase-2 plasma membrane localization and vascular endothelial cadherin binding because Src homology phosphatase-2 indirectly regulates dephosphorylation of Src tyrosine 527 that inhibits Src activity. Using immunohistochemistry and immunoprecipitation, we found that TXNIP prevented Src homology phosphatase-2–vascular endothelial cadherin interaction. Conclusions: In summary, these data characterize a fluid shear stress–mediated mechanism for stress fiber formation that involves a TXNIP-dependent vascular endothelial cadherin–Src homology phosphatase-2–Src pathway.

Thioredoxin-Interacting Protein Is a Biomechanical Regulator of Src ActivityNovelty and Significance

Rationale: Fluid shear stress differentially regulates endothelial cell stress fiber formation with decreased stress fibers in areas of disturbed flow compared with steady flow areas. Importantly, stress fibers are critical for several endothelial cell functions including cell shape, mechano-signal transduction, and endothelial cell–cell junction integrity. A key mediator of steady flow–induced stress fiber formation is Src that regulates downstream signaling mediators such as phosphorylation of cortactin, activity of focal adhesion kinase, and small GTPases. Objective: Previously, we showed that thioredoxin-interacting protein (TXNIP, also VDUP1 [vitamin D upregulated protein 1] and TBP-2 [thioredoxin binding protein 2]) was regulated by fluid shear stress; TXNIP expression was increased in disturbed flow compared with steady flow areas. Although TXNIP was originally characterized for its role in redox and metabolic cellular functions, recent reports show important scaffold functions related to its α-arrestin structure. Based on these findings, we hypothesized that TXNIP acts as a biomechanical sensor that regulates Src kinase activity and stress fiber formation. Methods and Results: Using en face immunohistochemistry of the aorta and cultured endothelial cells, we show inverse relationship between TXNIP expression and Src activity. Specifically, steady flow increased Src activity and stress fiber formation, whereas it decreased TXNIP expression. In contrast, disturbed flow had opposite effects. We studied the role of TXNIP in regulating Src homology phosphatase-2 plasma membrane localization and vascular endothelial cadherin binding because Src homology phosphatase-2 indirectly regulates dephosphorylation of Src tyrosine 527 that inhibits Src activity. Using immunohistochemistry and immunoprecipitation, we found that TXNIP prevented Src homology phosphatase-2–vascular endothelial cadherin interaction. Conclusions: In summary, these data characterize a fluid shear stress–mediated mechanism for stress fiber formation that involves a TXNIP-dependent vascular endothelial cadherin–Src homology phosphatase-2–Src pathway. # Novelty and Significance {#article-title-59}Rationale: Fluid shear stress differentially regulates endothelial cell stress fiber formation with decreased stress fibers in areas of disturbed flow compared with steady flow areas. Importantly, stress fibers are critical for several endothelial cell functions including cell shape, mechano-signal transduction, and endothelial cell–cell junction integrity. A key mediator of steady flow–induced stress fiber formation is Src that regulates downstream signaling mediators such as phosphorylation of cortactin, activity of focal adhesion kinase, and small GTPases. Objective: Previously, we showed that thioredoxin-interacting protein (TXNIP, also VDUP1 [vitamin D upregulated protein 1] and TBP-2 [thioredoxin binding protein 2]) was regulated by fluid shear stress; TXNIP expression was increased in disturbed flow compared with steady flow areas. Although TXNIP was originally characterized for its role in redox and metabolic cellular functions, recent reports show important scaffold functions related to its &agr;-arrestin structure. Based on these findings, we hypothesized that TXNIP acts as a biomechanical sensor that regulates Src kinase activity and stress fiber formation. Methods and Results: Using en face immunohistochemistry of the aorta and cultured endothelial cells, we show inverse relationship between TXNIP expression and Src activity. Specifically, steady flow increased Src activity and stress fiber formation, whereas it decreased TXNIP expression. In contrast, disturbed flow had opposite effects. We studied the role of TXNIP in regulating Src homology phosphatase-2 plasma membrane localization and vascular endothelial cadherin binding because Src homology phosphatase-2 indirectly regulates dephosphorylation of Src tyrosine 527 that inhibits Src activity. Using immunohistochemistry and immunoprecipitation, we found that TXNIP prevented Src homology phosphatase-2–vascular endothelial cadherin interaction. Conclusions: In summary, these data characterize a fluid shear stress–mediated mechanism for stress fiber formation that involves a TXNIP-dependent vascular endothelial cadherin–Src homology phosphatase-2–Src pathway.