An overview of CCN4 (WISP1) role in human diseases.

CCN4 (cellular communication network factor 4), a highly conserved, secreted cysteine-rich matricellular protein is emerging as a key player in the development and progression of numerous disease pathologies, including cancer, fibrosis, metabolic and inflammatory disorders. Over the past two decades, extensive research on CCN4 and its family members uncovered their diverse cellular mechanisms and biological functions, including but not limited to cell proliferation, migration, invasion, angiogenesis, wound healing, repair, and apoptosis. Recent studies have demonstrated that aberrant CCN4 expression and/or associated downstream signaling is key to a vast array of pathophysiological etiology, suggesting that CCN4 could be utilized not only as a non-invasive diagnostic or prognostic marker, but also as a promising therapeutic target. The cognate receptor of CCN4 remains elusive till date, which limits understanding of the mechanistic insights on CCN4 driven disease pathologies. However, as therapeutic agents directed against CCN4 begin to make their way into the clinic, that may start to change. Also, the pathophysiological significance of CCN4 remains underexplored, hence further research is needed to shed more light on its disease and/or tissue specific functions to better understand its clinical translational benefit. This review highlights the compelling evidence of overlapping and/or diverse functional and mechanisms regulated by CCN4, in addition to addressing the challenges, study limitations and knowledge gaps on CCN4 biology and its therapeutic potential.

Photoacoustic imaging of the dynamics of a dye-labeled IgG4 monoclonal antibody in subcutaneous tissue reveals a transient decrease in murine blood oxygenation under anesthesia.

Significance: Over 100 monoclonal antibodies have been approved by the U.S. Food and Drug Administration (FDA) for clinical use; however, a paucity of knowledge exists regarding the injection site behavior of these formulated therapeutics, particularly the effect of antibody, formulation, and tissue at the injection site. A deeper understanding of antibody behavior at the injection site, especially on blood oxygenation through imaging, will help design improved versions of the therapeutics for a wide range of diseases. Aim: The aim of this research is to understand the dynamics of monoclonal antibodies at the injection site as well as how the antibody itself affects the functional characteristics of the injection site [e.g., blood oxygen saturation (sO2)]. Approach: We employed triple-wavelength equipped functional photoacoustic imaging to study the dynamics of dye-labeled and unlabeled monoclonal antibodies at the site of injection in a mouse ear. We injected a near-infrared dye-labeled (and unlabeled) human IgG4 isotype control antibody into the subcutaneous space in mouse ears to analyze the injection site dynamics and quantify molecular movement, as well as its effect on local hemodynamics. Results: We performed pharmacokinetic studies of the antibody in different regions of the mouse body to show that dye labeling does not alter the pharmacokinetic characteristics of the antibody and that mouse ear is a viable model for these initial studies. We explored the movement of the antibody in the interstitial space to show that the bolus area grows by ∼300% over 24 h. We discovered that injection of the antibody transiently reduces the local sO2 levels in mice after prolonged anesthesia without affecting the total hemoglobin content and oxygen extraction fraction. Conclusions: This finding on local oxygen saturation opens a new avenue of study on the functional effects of monoclonal antibody injections. We also show the suitability of the mouse ear model to study antibody dynamics through high-resolution imaging techniques. We quantified the movement of antibodies at the injection site caused by the interstitial fluid, which could be helpful for designing antibodies with tailored absorption speeds in the future.

Characterization of a robust mouse model of heart failure with preserved ejection fraction.

Heart failure (HF) is a leading cause of morbidity and mortality particularly in older adults and patients with multiple metabolic comorbidities. Heart failure with preserved ejection fraction (HFpEF) is a clinical syndrome with multisystem organ dysfunction in which patients develop symptoms of HF as a result of high left ventricular (LV) diastolic pressure in the context of normal or near normal LV ejection fraction (LVEF; ≥50%). Challenges to create and reproduce a robust rodent phenotype that recapitulates the multiple comorbidities that exist in this syndrome explain the presence of various animal models that fail to satisfy all the criteria of HFpEF. Using a continuous infusion of angiotensin II and phenylephrine (ANG II/PE), we demonstrate a strong HFpEF phenotype satisfying major clinically relevant manifestations and criteria of this pathology, including exercise intolerance, pulmonary edema, concentric myocardial hypertrophy, diastolic dysfunction, histological signs of microvascular impairment, and fibrosis. Conventional echocardiographic analysis of diastolic dysfunction identified early stages of HFpEF development and speckle tracking echocardiography analysis including the left atrium (LA) identified strain abnormalities indicative of contraction-relaxation cycle impairment. Diastolic dysfunction was validated by retrograde cardiac catheterization and analysis of LV end-diastolic pressure (LVEDP). Among mice that developed HFpEF, two major subgroups were identified with predominantly perivascular fibrosis and interstitial myocardial fibrosis. In addition to major phenotypic criteria of HFpEF that were evident at early stages of this model (3 and 10 days), accompanying RNAseq data demonstrate activation of pathways associated with myocardial metabolic changes, inflammation, activation of extracellular matrix (ECM) deposition, microvascular rarefaction, and pressure- and volume-related myocardial stress.NEW & NOTEWORTHY Heart failure with preserved ejection fraction (HFpEF) is an emerging epidemic affecting up to half of patients with heart failure. Here we used a chronic angiotensin II/phenylephrine (ANG II/PE) infusion model and instituted an updated algorithm for HFpEF assessment. Given the simplicity in generating this model, it may become a useful tool for investigating pathogenic mechanisms, identification of diagnostic markers, and for drug discovery aimed at both prevention and treatment of HFpEF.

Long-Duration and Non-Invasive Photoacoustic Imaging of Multiple Anatomical Structures in a Live Mouse Using a Single Contrast Agent.

Long-duration in vivo simultaneous imaging of multiple anatomical structures is useful for understanding physiological aspects of diseases, informative for molecular optimization in preclinical models, and has potential applications in surgical settings to improve clinical outcomes. Previous studies involving simultaneous imaging of multiple anatomical structures, for example, blood and lymphatic vessels as well as peripheral nerves and sebaceous glands, have used genetically engineered mice, which require expensive and time-consuming methods. Here, an IgG4 isotype control antibody is labeled with a near-infrared dye and injected into a mouse ear to enable simultaneous visualization of blood and lymphatic vessels, peripheral nerves, and sebaceous glands for up to 3 h using photoacoustic microscopy. For multiple anatomical structure imaging, peripheral nerves and sebaceous glands are imaged inside the injected dye-labeled antibody mass while the lymphatic vessels are visualized outside the mass. The efficacy of the contrast agent to label and localize deep medial lymphatic vessels and lymph nodes using photoacoustic computed tomography is demonstrated. The capability of a single injectable contrast agent to image multiple structures for several hours will potentially improve preclinical therapeutic optimization, shorten discovery timelines, and enable clinical treatments.

Photoacoustic imaging reveals mechanisms of rapid-acting insulin formulations dynamics at the injection site.

OBJECTIVE: Ultra-rapid insulin formulations control postprandial hyperglycemia; however, inadequate understanding of injection site absorption mechanisms is limiting further advancement. We used photoacoustic imaging to investigate the injection site dynamics of dye-labeled insulin lispro in the Humalog® and Lyumjev® formulations using the murine ear cutaneous model and correlated it with results from unlabeled insulin lispro in pig subcutaneous injection model. METHODS: We employed dual-wavelength optical-resolution photoacoustic microscopy to study the absorption and diffusion of the near-infrared dye-labeled insulin lispro in the Humalog and Lyumjev formulations in mouse ears. We mathematically modeled the experimental data to calculate the absorption rate constants and diffusion coefficients. We studied the pharmacokinetics of the unlabeled insulin lispro in both the Humalog and Lyumjev formulations as well as a formulation lacking both the zinc and phenolic preservative in pigs. The association state of insulin lispro in each of the formulations was characterized using SV-AUC and NMR spectroscopy. RESULTS: Through experiments using murine and swine models, we show that the hexamer dissociation rate of insulin lispro is not the absorption rate-limiting step. We demonstrated that the excipients in the Lyumjev formulation produce local tissue expansion and speed both insulin diffusion and microvascular absorption. We also show that the diffusion of insulin lispro at the injection site drives its initial absorption; however, the rate at which the insulin lispro crosses the blood vessels is its overall absorption rate-limiting step. CONCLUSIONS: This study provides insights into injection site dynamics of insulin lispro and the impact of formulation excipients. It also demonstrates photoacoustic microscopy as a promising tool for studying protein therapeutics. The results from this study address critical questions around the subcutaneous behavior of insulin lispro and the formulation excipients, which could be useful to make faster and better controlled insulin formulations in the future.

Targeting VE-PTP phosphatase protects the kidney from diabetic injury.

Diabetic nephropathy is a leading cause of end-stage kidney failure. Reduced angiopoietin-TIE2 receptor tyrosine kinase signaling in the vasculature leads to increased vascular permeability, inflammation, and endothelial cell loss and is associated with the development of diabetic complications. Here, we identified a mechanism to explain how TIE2 signaling is attenuated in diabetic animals. Expression of vascular endothelial protein tyrosine phosphatase VE-PTP (also known as PTPRB), which dephosphorylates TIE2, is robustly up-regulated in the renal microvasculature of diabetic rodents, thereby reducing TIE2 activity. Increased VE-PTP expression was dependent on hypoxia-inducible factor transcriptional activity in vivo. Genetic deletion of VE-PTP restored TIE2 activity independent of ligand availability and protected kidney structure and function in a mouse model of severe diabetic nephropathy. Mechanistically, inhibition of VE-PTP activated endothelial nitric oxide synthase and led to nuclear exclusion of the FOXO1 transcription factor, reducing expression of pro-inflammatory and pro-fibrotic gene targets. In sum, we identify inhibition of VE-PTP as a promising therapeutic target to protect the kidney from diabetic injury.

VEGF expands erythropoiesis via hypoxia-independent induction of erythropoietin in noncanonical perivascular stromal cells.

Insufficient erythropoiesis due to increased demand is usually met by hypoxia-driven up-regulation of erythropoietin (Epo). Here, we uncovered vascular endothelial growth factor (VEGF) as a novel inducer of Epo capable of increasing circulating Epo under normoxic, nonanemic conditions in a previously unrecognized reservoir of Epo-producing cells (EPCs), leading to expansion of the erythroid progenitor pool and robust splenic erythropoiesis. Epo induction by VEGF occurs in kidney, liver, and spleen in a population of Gli1+SMA+PDGFRß+ cells, a signature shared with vascular smooth muscle cells (VSMCs) derived from mesenchymal stem cell-like progenitors. Surprisingly, inhibition of PDGFRß signaling, but not VEGF signaling, abrogated VEGF-induced Epo synthesis. We thus introduce VEGF as a new player in Epo induction and perivascular Gli1+SMA+PDGFRß+ cells as a previously unrecognized EPC reservoir that could be harnessed for augmenting Epo synthesis in circumstances such as chronic kidney disease where production by canonical EPCs is compromised.

Impaired tumor growth and angiogenesis in mice heterozygous for Vegfr2 (Flk1).

VEGF signaling through its tyrosine kinase receptor, VEGFR2 (FLK1), is critical for tumor angiogenesis. Previous studies have identified a critical gene dosage effect of VegfA in embryonic development and vessel homeostasis, neovascularization, and tumor growth, and potent inhibitors of VEGFR2 have been used to treat a variety of cancers. Inhibition of FGFR signaling has also been considered as an antiangiogenic approach to treat a variety of cancers. Inhibition of VEGFR2 with neutralizing antibodies or with pharmacological inhibitors of the VEGFR tyrosine kinase domain has at least short-term efficacy with some cancers; however, also affects vessel homeostasis, leading to adverse complications. We investigate gene dosage effects of Vegfr2, Fgfr1, and Fgfr2 in three independent mouse models of tumorigenesis: two-stage skin chemical carcinogenesis, and sub-cutaneous transplantation of B16F0 melanoma and Lewis Lung Carcinoma (LLC). Mice heterozygous for Vegfr2 display profound defects in supporting tumor growth and angiogenesis. Unexpectedly, additional deletion of endothelial Fgfr1 and Fgfr2 in Vegfr2 heterozygous mice shows similar tumor growth and angiogenesis as the Vegfr2 heterozygous mice. Notably, hematopoietic deletion of two alleles of Vegfr2 had minimal impact on tumor growth, with little effect on angiogenesis, reinforcing the importance of endothelial Vegfr2 heterozygosity. These studies reveal previously unrecognized Vegfr2 gene dosage effects in tumor angiogenesis and a lack of synergy between VEGFR2 and endothelial FGFR1/2 signaling during tumor growth.

Injury-Mediated Vascular Regeneration Requires Endothelial ER71/ETV2.

OBJECTIVE: Comprehensive understanding of the mechanisms regulating angiogenesis might provide new strategies for angiogenic therapies for treating diverse physiological and pathological ischemic conditions. The E-twenty six (ETS) factor Ets variant 2 (ETV2; aka Ets-related protein 71) is essential for the formation of hematopoietic and vascular systems. Despite its indispensable function in vessel development, ETV2 role in adult angiogenesis has not yet been addressed. We have therefore investigated the role of ETV2 in vascular regeneration. APPROACH AND RESULTS: We used endothelial Etv2 conditional knockout mice and ischemic injury models to assess the role of ETV2 in vascular regeneration. Although Etv2 expression was not detectable under steady-state conditions, its expression was readily observed in endothelial cells after injury. Mice lacking endothelial Etv2 displayed impaired neovascularization in response to eye injury, wounding, or hindlimb ischemic injury. Lentiviral Etv2 expression in ischemic hindlimbs led to improved recovery of blood perfusion with enhanced vessel formation. After injury, fetal liver kinase 1 (Flk1), aka VEGFR2, expression and neovascularization were significantly upregulated by Etv2, whereas Flk1 expression and vascular endothelial growth factor response were significantly blunted in Etv2-deficient endothelial cells. Conversely, enforced Etv2 expression enhanced vascular endothelial growth factor-mediated endothelial sprouting from embryoid bodies. Lentiviral Flk1 expression rescued angiogenesis defects in endothelial Etv2 conditional knockout mice after hindlimb ischemic injury. Furthermore, Etv2(+/-); Flk1(+/-) double heterozygous mice displayed a more severe hindlimb ischemic injury response compared with Etv2(+/-) or Flk1(+/-) heterozygous mice, revealing an epistatic interaction between ETV2 and FLK1 in vascular regeneration. CONCLUSIONS: Our study demonstrates a novel obligatory role for the ETV2 in postnatal vascular repair and regeneration.

Endothelial cell FGF signaling is required for injury response but not for vascular homeostasis.

Endothelial cells (ECs) express fibroblast growth factor receptors (FGFRs) and are exquisitely sensitive to FGF signals. However, whether the EC or another vascular cell type requires FGF signaling during development, homeostasis, and response to injury is not known. Here, we show that Flk1-Cre or Tie2-Cre mediated deletion of FGFR1 and FGFR2 (Fgfr1/2(Flk1-Cre) or Fgfr1/2(Tie2-Cre) mice), which results in deletion in endothelial and hematopoietic cells, is compatible with normal embryonic development. As adults, Fgfr1/2(Flk1-Cre) mice maintain normal blood pressure and vascular reactivity and integrity under homeostatic conditions. However, neovascularization after skin or eye injury was significantly impaired in both Fgfr1/2(Flk1-Cre) and Fgfr1/2(Tie2-Cre) mice, independent of either hematopoietic cell loss of FGFR1/2 or vascular endothelial growth factor receptor 2 (Vegfr2) haploinsufficiency. Also, impaired neovascularization was associated with delayed cutaneous wound healing. These findings reveal a key requirement for cell-autonomous EC FGFR signaling in injury-induced angiogenesis, but not for vascular homeostasis, identifying the EC FGFR signaling pathway as a target for diseases associated with aberrant vascular proliferation, such as age-related macular degeneration, and for modulating wound healing without the potential toxicity associated with direct manipulation of systemic FGF or VEGF activity.

Conditional HIF-1 induction produces multistage neovascularization with stage-specific sensitivity to VEGFR inhibitors and myeloid cell independence.

Neovascularization is a crucial component of tumor growth and ischemia. Although prior work primarily used disease models, delineation of neovascularization in the absence of disease can reveal intrinsic mechanisms of microvessel regulation amenable to manipulation in illness. We created a conditional model of epithelial HIF-1 induction in adult mice (TetON-HIF-1 mice). Longitudinal photoacoustic microscopy (L-PAM) was coincidentally developed for noninvasive, label-free serial imaging of red blood cell-perfused vasculature in the same mouse for weeks to months. TetON-HIF-1 mice evidenced 3 stages of neovascularization: development, maintenance, and transgene-dependent regression. Regression occurred despite extensive and tight pericyte coverage. L-PAM mapped microvascular architecture and quantified volumetric changes in neocapillary morphogenesis, arteriovenous remodeling, and microvessel regression. Developmental stage endothelial proliferation down-regulation was associated with a DNA damage checkpoint consisting of p53, p21, and endothelial γ-H2AX induction. The neovasculature was temporally responsive to VEGFR2 immuno-blockade, with the developmental stage sensitive, and the maintenance stage resistant, to DC101 treatment. L-PAM analysis also pinpointed microvessels ablated or resistant to VEGFR2 immuno-blockade. HIF-1-recruited myeloid cells did not mediate VEGFR2 inhibitor resistance. Thus, HIF-1 neovascularization in the absence of disease is self-regulated via cell autonomous endothelial checkpoints, and resistant to angiogenesis inhibitors independent of myeloid cells.