Power-Doppler Ultrasonic Imaging of Peripheral Perfusion in Diabetic Mice.

OBJECTIVE: We explored the capabilities of power-Doppler ultrasonic (PD-US) imaging without contrast enhancement for monitoring changes in muscle perfusion over time. METHODS: Ischemic recovery was observed in healthy and type II diabetic male and female mice with and without exercise. In separate studies, perfusion was measured during and after 5-min ischemic periods and during four-week recovery periods following irreversible femoral ligation. A goal was to assess how well PD-US estimates tracked the diabetic-related changes in endothelial function that influenced perfusion. RESULTS: The average perfusion recovery time following femoral ligation increased 47% in diabetic males and 74% in diabetic females compared with non-diabetic mice. Flow-mediated dilation in conduit arteries and the reactive hyperemia index in resistive vessels each declined by one half in sedentary diabetic mice compared with sedentary non-diabetic mice. We found that exercise reduced the loss of endothelial function from diabetes in both sexes. The reproducibility of perfusion measurements was limited primarily by our ability to select the same region in muscle and to effectively filter tissue clutter. CONCLUSIONS/SIGNIFICANCE: PD-US measurements can precisely follow site-specific changes in skeletal muscle perfusion related to diabetes over time, which fills the need for techniques capable of regularly monitoring atherosclerotic changes leading to ischemic vascular pathologies.

The Impact of LY487379 or CDPPB on eNOS Expression in the Mouse Brain and the Effect of Joint Administration of Compounds with NO• Releasers on MK-801- or Scopolamine-Driven Cognitive Dysfunction in Mice.

The role of endothelial nitric oxide synthase (eNOS) in the regulation of a variety of biological processes is well established, and its dysfunction contributes to brain pathologies, including schizophrenia or Alzheimer's disease (AD). Positive allosteric modulators (PAMs) of metabotropic glutamate (mGlu) receptors were shown to be effective procognitive compounds, but little is known about their impact on eNOS expression and stability. Here, we investigated the influence of the acute and chronic administration of LY487379 or CDPPB (mGlu2 and mGlu5 PAMs), on eNOS expression in the mouse brain and the effect of the joint administration of the ligands with nitric oxide (NO) releasers, spermineNONOate or DETANONOate, in different combinations of doses, on MK-801- or scopolamine-induced amnesia in the novel object recognition (NOR) test. Our results indicate that both compounds provoked eNOS monomer formation, and CDPPB at a dose of 5 mg/kg exaggerated the effect of MK-801 or scopolamine. The coadministration of spermineNONOate or DETANONOate enhanced the antiamnesic effect of CDPPB or LY487379. The best activity was observed for ineffective or moderate dose combinations. The results indicate that treatment with mGluR2 and mGluR5 PAMs may be burdened with the risk of promoting eNOS uncoupling through the induction of dimer dissociation. Administration of the lowest possible doses of the compounds with NO• donors, which themselves have procognitive efficacy, may be proposed for the treatment of schizophrenia or AD.

Receptor for advanced glycation end-products: Biological significance and imaging applications.

The receptor for advanced glycation end-products (RAGE or AGER) is a transmembrane, immunoglobulin-like receptor that, due to its multiple isoform structures, binds to a diverse range of endo- and exogenous ligands. RAGE activation caused by the ligand binding initiates a cascade of complex pathways associated with producing free radicals, such as reactive nitric oxide and oxygen species, cell proliferation, and immunoinflammatory processes. The involvement of RAGE in the pathogenesis of disorders such as diabetes, inflammation, tumor progression, and endothelial dysfunction is dictated by the accumulation of advanced glycation end-products (AGEs) at pathologic states leading to sustained RAGE upregulation. The involvement of RAGE and its ligands in numerous pathologies and diseases makes RAGE an interesting target for therapy focused on the modulation of both RAGE expression or activation and the production or exogenous administration of AGEs. Despite the known role that the RAGE/AGE axis plays in multiple disease states, there remains an urgent need to develop noninvasive, molecular imaging approaches that can accurately quantify RAGE levels in vivo that will aid in the validation of RAGE and its ligands as biomarkers and therapeutic targets. This article is categorized under: Diagnostic Tools > In Vivo Nanodiagnostics and Imaging Diagnostic Tools > Biosensing.

Coordination Chemistry of Sulfur-Containing Bifunctional Chelators: Toward in Vivo Stabilization of 64Cu PET Imaging Agents for Alzheimer's Disease.

The broader utilization of 64Cu positron emission tomography (PET) imaging agents has been hindered by the unproductive demetalation induced by bioreductants. To advance the development of 64Cu-based PET imaging tracers for Alzheimer's Disease (AD), there is a need for novel ligand design strategies. In this study, we developed sulfur-containing dithiapyridinophane (N2S2) bifunctional chelators (BFCs) as well as all nitrogen-based diazapyridinophane (N4) BFCs to compare their abilities to chelate Cu and target Aß aggregates. Through spectrophotometric titrations and electrochemical measurements, we have demonstrated that the N2S2-based BFCs exhibit >10 orders of magnitude higher binding affinity toward Cu(I) compared to their N4-based counterparts, while both types of BFCs exhibit high stability constants toward Cu(II). Notably, solid state structures for both Cu(II) and Cu(I) complexes supported by the two ligand frameworks were obtained, providing molecular insights into their copper chelating abilities. Aß binding experiments were conducted to study the structure-affinity relationship, and fluorescence microscopy imaging studies confirmed the selective labeling of the BFCs and their copper complexes. Furthermore, we investigated the potential of these ligands for the 64Cu-based PET imaging of AD through radiolabeling and autoradiography studies. We believe our findings provide molecular insights into the design of bifunctional Cu chelators that can effectively stabilize both Cu(II) and Cu(I) and, thus, can have significant implications for the development of 64Cu PET imaging as a diagnostic tool for AD.

In Vitro and In Vivo Imaging-Based Evaluation of Doxorubicin Anticancer Treatment in Combination with the Herbal Medicine Black Cohosh.

As a substitution for hormone replacement therapy, many breast cancer patients use black cohosh (BC) extracts in combination with doxorubicin (DOX)-based chemotherapy. In this study, we evaluated the viability and survival of BC- and DOX-treated MCF-7 cells. A preclinical model of MCF-7 xenografts was used to determine the influence of BC and DOX administration on tumor growth and metabolism. The number of apoptotic cells after incubation with both DOX and BC was significantly increased (~100%) compared to the control. Treatment with DOX altered the potential of MCF-7 cells to form colonies; however, coincubation with BC did not affect this process. In vivo, PET-CT imaging showed that combined treatment of DOX and BC induced a significant reduction in both metabolic activity (29%) and angiogenesis (32%). Both DOX and BC treatments inhibited tumor growth by 20% and 12%, respectively, and combined by 57%, vs. control. We successfully demonstrated that BC increases cytotoxic effects of DOX, resulting in a significant reduction in tumor size. Further studies regarding drug transport and tumor growth biomarkers are necessary to establish the underlying mechanism and potential clinical use of BC in breast cancer patients.

RAGE as a Novel Biomarker for Prostate Cancer: A Systematic Review and Meta-Analysis.

The receptor for advanced glycation end-products (RAGE) has been implicated in driving prostate cancer (PCa) growth, aggression, and metastasis through the fueling of chronic inflammation in the tumor microenvironment. This systematic review and meta-analysis summarizes and analyzes the current clinical and preclinical data to provide insight into the relationships among RAGE levels and PCa, cancer grade, and molecular effects. A multi-database search was used to identify original clinical and preclinical research articles examining RAGE expression in PCa. After screening and review, nine clinical and six preclinical articles were included. The associations of RAGE differentiating benign prostate hyperplasia (BPH) or normal prostate from PCa and between tumor grades were estimated using odds ratios (ORs) and associated 95% confidence intervals (CI). Pooled estimates were calculated using random-effect models due to study heterogeneity. The clinical meta-analysis found that RAGE expression was highly likely to be increased in PCa when compared to BPH or normal prostate (OR: 11.3; 95% CI: 4.4-29.1) and that RAGE was overexpressed in high-grade PCa when compared to low-grade PCa (OR: 2.5; 95% CI: 1.8-3.4). In addition, meta-analysis estimates of preclinical studies performed by albatross plot generation found robustly positive associations among RAGE expression/activation and PCa growth and metastatic potential. This review demonstrates that RAGE expression is strongly tied to PCa progression and can serve as an effective diagnostic target to differentiate between healthy prostate, low-grade PCa, and high-grade PCa, with potential theragnostic applications.

Amphiphilic Molecules Exhibiting Zwitterionic Excited-State Intramolecular Proton Transfer and Near-Infrared Emission for the Detection of Amyloid ß Aggregates in Alzheimer's Disease.

Chromophores with zwitterionic excited-state intramolecular proton transfer (ESIPT) have been shown to have larger Stock shifts and red-shifted emission wavelengths compared to the conventional π-delocalized ESIPT molecules. However, there is still a dearth of design strategies to expand the current library of zwitterionic ESIPT compounds. Herein, a novel zwitterionic excited-state intramolecular proton transfer system is reported, enabled by addition of 1,4,7-triazacyclononane (TACN) fragments on a dicyanomethylene-4H-pyran (DCM) scaffold. The solvent-dependent steady-state photophysical studies, pKa measurements, and computational analysis strongly support that the ESIPT process is more efficient with two TACN groups attached to the DCM scaffold and not affected by polar protic solvents. Impressively, compound DCM-OH-2-DT exhibits a near-infrared (NIR) emission at 740 nm along with an uncommonly large Stokes shift. Moreover, DCM-OH-2-DT shows high affinity towards soluble amyloid ß (Aß) oligomers in vitro and in 5xFAD mouse brain sections, and we have successfully applied DCM-OH-2-DT for the in vivo imaging of Aß aggregates and demonstrated its potential use as an early diagnostic agent for AD. Overall, this study can provide a general molecular design strategy for developing new zwitterionic ESIPT compounds with NIR emission in vivo imaging applications.

Endothelial dysfunction due to eNOS uncoupling: molecular mechanisms as potential therapeutic targets.

Nitric oxide (NO) is one of the most important molecules released by endothelial cells, and its antiatherogenic properties support cardiovascular homeostasis. Diminished NO bioavailability is a common hallmark of endothelial dysfunction underlying the pathogenesis of the cardiovascular disease. Vascular NO is synthesized by endothelial nitric oxide synthase (eNOS) from the substrate L-arginine (L-Arg), with tetrahydrobiopterin (BH4) as an essential cofactor. Cardiovascular risk factors such as diabetes, dyslipidemia, hypertension, aging, or smoking increase vascular oxidative stress that strongly affects eNOS activity and leads to eNOS uncoupling. Uncoupled eNOS produces superoxide anion (O2-) instead of NO, thus becoming a source of harmful free radicals exacerbating the oxidative stress further. eNOS uncoupling is thought to be one of the major underlying causes of endothelial dysfunction observed in the pathogenesis of vascular diseases. Here, we discuss the main mechanisms of eNOS uncoupling, including oxidative depletion of the critical eNOS cofactor BH4, deficiency of eNOS substrate L-Arg, or accumulation of its analog asymmetrical dimethylarginine (ADMA), and eNOS S-glutathionylation. Moreover, potential therapeutic approaches that prevent eNOS uncoupling by improving cofactor availability, restoration of L-Arg/ADMA ratio, or modulation of eNOS S-glutathionylation are briefly outlined.

Molecular Imaging and Nanotechnology-Emerging Tools in Diagnostics and Therapy.

Personalized medicine is emerging as a new goal in the diagnosis and treatment of diseases. This approach aims to establish differences between patients suffering from the same disease, which allows to choose the most effective treatment. Molecular imaging (MI) enables advanced insight into molecule interactions and disease pathology, improving the process of diagnosis and therapy and, for that reason, plays a crucial role in personalized medicine. Nanoparticles are widely used in MI techniques due to their size, high surface area to volume ratio, and multifunctional properties. After conjugation to specific ligands and drugs, nanoparticles can transport therapeutic compounds directly to their area of action and therefore may be used in theranostics-the simultaneous implementation of treatment and diagnostics. This review summarizes different MI techniques, including optical imaging, ultrasound imaging, magnetic resonance imaging, nuclear imaging, and computed tomography imaging with theranostics nanoparticles. Furthermore, it explores the potential use of constructs that enables multimodal imaging and track diseases in real time.

Dextran-Mimetic Quantum Dots for Multimodal Macrophage Imaging In Vivo, Ex Vivo, and In Situ.

Macrophages are white blood cells with diverse functions contributing to a healthy immune response as well as the pathogenesis of cancer, osteoarthritis, atherosclerosis, and obesity. Due to their pleiotropic and dynamic nature, tools for imaging and tracking these cells at scales spanning the whole body down to microns could help to understand their role in disease states. Here we report fluorescent and radioisotopic quantum dots (QDs) for multimodal imaging of macrophage cells in vivo, ex vivo, and in situ. Macrophage specificity is imparted by click-conjugation to dextran, a biocompatible polysaccharide that natively targets these cell types. The emission spectral band of the crystalline semiconductor core was tuned to the near-infrared for optical imaging deep in tissue, and probes were covalently conjugated to radioactive iodine for nuclear imaging. The performance of these probes was compared with all-organic dextran probe analogues in terms of their capacity to target macrophages in visceral adipose tissue using in vivo positron emission tomography/computed tomography (PET/CT) imaging, in vivo fluorescence imaging, ex vivo fluorescence, post-mortem isotopic analyses, and optical microscopy. All probe classes exhibited equivalent physicochemical characteristics in aqueous solution and similar in vivo targeting specificity. However, dextran-mimetic QDs provided enhanced signal-to-noise ratio for improved optical quantification, long-term photostability, and resistance to chemical fixation. In addition, the vascular circulation time for the QD-based probes was extended 9-fold compared with dextran, likely due to differences in conformational flexibility. The enhanced photophysical and photochemical properties of dextran-mimetic QDs may accelerate applications in macrophage targeting, tracking, and imaging across broad resolution scales, particularly advancing capabilities in single-cell and single-molecule imaging and quantification.

Molecularly targeted nanoparticles: an emerging tool for evaluation of expression of the receptor for advanced glycation end products in a murine model of peripheral artery disease.

BACKGROUND: Molecular imaging with molecularly targeted probes is a powerful tool for studying the spatio-temporal interactions between complex biological processes. The pivotal role of the receptor for advanced glycation end products (RAGE), and its involvement in numerous pathological processes, aroused the demand for RAGE-targeted imaging in various diseases. In the present study, we evaluated the use of a diagnostic imaging agent for RAGE quantification in an animal model of peripheral artery disease, a multimodal dual-labeled probe targeted at RAGE (MMIA-CML). METHODS: PAMAM dendrimer was conjugated with Nε-carboxymethyl-lysine (CML) modified albumin to synthesize the RAGE-targeted probe. A control untargeted agent carried native non-modified human albumin (HSA). Bifunctional p-SCN-Bn-NOTA was used to conjugate the 64Cu radioisotope. Surgical right femoral artery ligation was performed on C57BL/6 male mice. One week after femoral artery ligation, mice were injected with MMIA-CML or MMIA-HSA labeled with 64Cu radioisotope and 60 min later in vivo microPET-CT imaging was performed. Immediately after PET imaging studies, the murine hindlimb muscle tissues were excised and prepared for gene and protein expression analysis. RAGE gene and protein expression was assessed using real-time qPCR and Western blot technique respectively. To visualize RAGE expression in excised tissues, microscopic fluorescence imaging was performed using RAGE-specific antibodies and RAGE-targeted and -control MMIA. RESULTS: Animals subjected to PET imaging exhibited greater MMIA-CML uptake in ischemic hindlimbs than non-ischemic hindlimbs. We observed a high correlation between fluorescent signal detection and radioactivity measurement. Significant RAGE gene and protein overexpression were observed in ischemic hindlimbs compared to non-ischemic hindlimbs at one week after surgical ligation. Fluorescence microscopic staining revealed significantly increased uptake of RAGE-targeted nanoparticles in both ischemic and non-ischemic muscle tissues compared to the control probe but at a higher level in ischemic hindlimbs. Ischemic tissue exhibited explicit RAGE dyeing following anti-RAGE antibody and high colocalization with the MMIA-CML targeted at RAGE. CONCLUSIONS: The present results indicate increased expression of RAGE in the ischemic hindlimb and enable the use of multimodal nanoparticles in both in vitro and in vivo experimental models, creating the possibility for imaging structural and functional changes with a RAGE-targeted tracer.

Pathogenesis of psoriasis in the "omic" era. Part IV. Epidemiology, genetics, immunopathogenesis, clinical manifestation and treatment of psoriatic arthritis.

Psoriatic arthritis (PsA) is a chronic, progressive, inflammatory arthropathy associated with psoriasis as well as a complex pathogenesis. Genetic and environmental factors trigger the development of the immune-mediated auto-inflammatory response in different sites: skin, bone marrow, entheses and synovial tissues. Studies of the last two decades have changed the view of PsA from a mild, non-progressive arthritis to an inflammatory systemic disease with serious health consequences, not only associated with joint dysfunction, but also with an increased risk of cardiovascular disease and socioeconomic consequences with significantly reduced quality of life. The joint damage starts early in the course of the disease, thus early recognition and treatment with modern biological treatments, which may modify the natural history and slow down progression of this debilitating disease, is essential for the patient long-term outcome.

Pathogenesis of psoriasis in the "omic" era. Part III. Metabolic disorders, metabolomics, nutrigenomics in psoriasis.

Psoriasis is a systemic disease that is strictly connected with metabolic disorders (insulin resistance, atherogenic dyslipidemia, arterial hypertension, and cardiovascular diseases). It occurs more often in patients with a more severe course of the disease. Obesity is specially an independent risk factor and it is associated with a worse treatment outcome because of the high inflammatory activity of visceral fatty tissue and the production of inflammatory mediators involved in the development of both psoriasis and metabolic disorders. However, in psoriasis the activation of the Th17/IL-17 and the abnormalities in the Th17/Treg balance axis are observed, but this pathomechanism does not fully explain the frequent occurrence of metabolic disorders. Therefore, there is a need to look for better biomarkers in the diagnosis, prognosis and monitoring of concomitant disorders and therapeutic effects in psoriasis. In addition, the education on the use of a proper diet as a prophylaxis for the development of the above disorders is an important element of holistic care for a patient with psoriasis. Diet may affect gene expression due to epigenetic modification which encompasses interactions of environment, nutrition and diseases. Patients with psoriasis should be advised to adopt proper diet and dietician support.

Intratumoral generation of photothermal gold nanoparticles through a vectorized biomineralization of ionic gold.

Various cancer cells have been demonstrated to have the capacity to form plasmonic gold nanoparticles when chloroauric acid is introduced to their cellular microenvironment. But their biomedical applications are limited, particularly considering the millimolar concentrations and longer incubation period of ionic gold. Here, we describe a simplistic method of intracellular biomineralization to produce plasmonic gold nanoparticles at micromolar concentrations within 30 min of application utilizing polyethylene glycol as delivery vector for ionic gold. We have characterized this process for intracellular gold nanoparticle formation, which progressively accumulates proteins as the ionic gold clusters migrate to the nucleus. This nano-vectorized application of ionic gold emphasizes its potential biomedical opportunities while reducing the quantity of ionic gold and required incubation time. To demonstrate its biomedical potential, we further induce in-situ biosynthesis of gold nanoparticles within MCF7 tumor mouse xenografts which is followed by its photothermal remediation.

Pathogenesis of psoriasis in the "omic" era. Part II. Genetic, genomic and epigenetic changes in psoriasis.

Psoriasis is a multifactorial disease in which genetic, environmental and epigenetic factors regulating gene expression play a key role. In the "genomic era", genome-wide association studies together with target genotyping platforms performed in different ethnic populations have found more than 50 genetic susceptible markers associated with the risk of psoriasis which have been identified so far. Up till now, the strongest association with the risk of the disease has been proved for HLA-C*06 gene. The majority of other psoriasis risk SNPs are situated near the genes encoding molecules involved in adaptive and innate immunity, and skin barrier function. Many contemporary studies indicate that the epigenetic changes: histone modification, promoter methylations, long non-coding and micro-RNA hyperexpression are considered as factors contributing to psoriasis pathogenesis as they regulate abnormal keratinocyte differentiation and proliferation, aberrant keratinocytes - inflammatory cells communication, neoangiogenesis and chronic inflammation. The circulating miRNAs detected in the blood may become specific markers in the diagnosis, prognosis and response to the treatment of the disease. The inhibition of expression in selected miRNAs may be a new promising therapy option for patients with psoriasis.

Pathogenesis of psoriasis in the "omic" era. Part I. Epidemiology, clinical manifestation, immunological and neuroendocrine disturbances.

Psoriasis is a common, chronic, inflammatory, immune-mediated skin disease affecting about 2% of the world's population. According to current knowledge, psoriasis is a complex disease that involves various genes and environmental factors, such as stress, injuries, infections and certain medications. The chronic inflammation of psoriasis lesions develops upon epidermal infiltration, activation, and expansion of type 1 and type 17 Th cells. Despite the enormous progress in understanding the mechanisms that cause psoriasis, the target cells and antigens that drive pathogenic T cell responses in psoriatic lesions are still unproven and the autoimmune basis of psoriasis still remains hypothetical. However, since the identification of the Th17 cell subset, the IL-23/Th17 immune axis has been considered a key driver of psoriatic inflammation, which has led to the development of biologic agents that target crucial elements of this pathway. Here we present the current understanding of various aspects in psoriasis pathogenesis.

Quantitative imaging of the receptor for advanced glycation end-products in prostate cancer.

PURPOSE: Current screening and monitoring of prostate cancer (PCa) is insufficient, producing inaccurate diagnoses. Presence of the receptor for advanced glycation end-products (RAGE) is associated with signature characteristics of PCa development such as cell proliferation, anchorage-independent growth, angiogenesis, migration, invasion, and poor patient survival. Therefore, we developed a preclinical multimodal imaging strategy targeted at RAGE to diagnose and monitor PCa. METHODS: In this work, RAGE-targeted multimodal nanoparticles (64Cu-Cy5-G4-CML) were synthesized and rendered functional for nuclear and optical imaging using previously established methods. The probe's binding affinity and targeting specificity was assessed in androgen-dependent (LNCaP) and androgen-independent (DU145) prostate cancer cells using flow cytometry and confocal microscopy. In vivo PET-CT imaging was used to evaluate RAGE levels in DU145 and LNCaP xenograft models in mice. Then, tumors were excised post-imaging for histological staining and autoradiography to further assess RAGE levels and targeting efficiency of the tracer. Finally, RAGE levels from human PCa samples of varying Gleason Scores were evaluated using Western blot and immunohistochemical staining. RESULTS: PCa cell culture studies confirmed adequate RAGE-targeting with 64Cu-Cy5-G4-CML with KD between 360 and 540 nM as measured by flow cytometry. In vivo PET-CT images of PCa xenografts revealed favorable kinetics, rapid blood clearance, and a non-homogenous, enhanced uptake in tumors, which varied based on cell type and tumor size with mean uptake between 0.5 and 1.4%ID/g. RAGE quantification of human samples confirmed increased RAGE uptake corresponding to increased Gleason scoring. CONCLUSIONS: Our study has shown that RAGE-targeted cancer imaging is feasible and could significantly impact PCa management.

Aberrant Expression of a Non-muscle RBFOX2 Isoform Triggers Cardiac Conduction Defects in Myotonic Dystrophy.

Myotonic dystrophy type 1 (DM1) is a multisystemic genetic disorder caused by the CTG repeat expansion in the 3'-untranslated region of DMPK gene. Heart dysfunctions occur in ∼80% of DM1 patients and are the second leading cause of DM1-related deaths. Herein, we report that upregulation of a non-muscle splice isoform of RNA-binding protein RBFOX2 in DM1 heart tissue-due to altered splicing factor and microRNA activities-induces cardiac conduction defects in DM1 individuals. Mice engineered to express the non-muscle RBFOX240 isoform in heart via tetracycline-inducible transgenesis, or CRISPR/Cas9-mediated genome editing, reproduced DM1-related cardiac conduction delay and spontaneous episodes of arrhythmia. Further, by integrating RNA binding with cardiac transcriptome datasets from DM1 patients and mice expressing the non-muscle RBFOX2 isoform, we identified RBFOX240-driven splicing defects in voltage-gated sodium and potassium channels, which alter their electrophysiological properties. Thus, our results uncover a trans-dominant role for an aberrantly expressed RBFOX240 isoform in DM1 cardiac pathogenesis.

Imaging the Landmarks of Vascular Recovery.

Background: Peripheral arterial disease (PAD) is a major worldwide health concern. Since the late 1990s therapeutic angiogenesis has been investigated as an alternative to traditional PAD treatments. Although positive preclinical results abound in the literature, the outcomes of human clinical trials have been discouraging. Among the challenges the field has faced has been a lack of standardization of the timings and measures used to validate new treatment approaches. Methods: In order to study the spatiotemporal dynamics of both perfusion and neovascularization in mice subjected to surgically-induced hindlimb ischemia (n= 30), we employed three label-free imaging modalities (a novel high-sensitivity ultrasonic Power Doppler methodology, laser speckle contrast, and photoacoustic imaging), as well as a tandem of radio-labeled molecular probes, 99mTc-NC100692 and 99mTc-BRU-5921 respectively, designed to detect two key modulators of angiogenic activity, αVß3 and HIF-1α , via scintigraphic imaging. Results: The multimodal imaging strategy reveals a set of "landmarks"-key physiological and molecular events in the healing process-that can serve as a standardized framework for describing the impact of emerging PAD treatments. These landmarks span the entire process of neovascularization, beginning with the rapid decreases in perfusion and oxygenation associated with ligation surgery, extending through pro-angiogenic changes in gene expression driven by the master regulator HIF-1α , and ultimately leading to complete functional revascularization of the affected tissues. Conclusions: This study represents an important step in the development of multimodal non-invasive imaging strategies for vascular research; the combined results offer more insight than can be gleaned through any of the individual imaging methods alone. Researchers adopting similar imaging strategies and will be better able to describe changes in the onset, duration, and strength of each of the landmarks of vascular recovery, yielding greater biological insight, and enabling more comprehensive cross-study comparisons. Perhaps most important, this study paves the road for more efficient translation of PAD research; emerging experimental treatments can be more effectively assessed and refined at the preclinical stage, ultimately leading to better next-generation therapies.