Vascular Signature Research Articles

Reduction in retinal microvascular perfusion during migraine attacks.

To determine if there are changes in structure and function of the retinal vasculature during and between migraine attacks using optical coherence tomography angiography (OCTA). Migraine attacks commonly include visual symptoms, but the potential role of the retina in these symptoms is not well understood. OCTA is a rapid, non-invasive imaging technique that is used to visualize the retinal microvasculature with high spatial resolution in a clinical setting. In this study we used OCTA to quantify different features of the retinal vasculature in patients with migraine during and between attacks, as well as in healthy controls (HCs). We performed a prospective cohort study of 37 patients with migraine with aura (MA) (median [interquartile range, IQR] age of 37 [14] years, 86% female) and 30with migraine without aura (MO) (median [IQR] age of 37 [17] years, 77% female) and 20 HCs (median [IQR] age of 35 [7] years, 50% female). Macular OCTA scans were obtained for all participants for the interictal analysis. In 12 MA and eight MO, scans were captured both during and outside of migraine attacks and five HCs had initial and repeat scans. In addition to analyzing the morphology of the foveal avascular zone, we calculated the vessel flux index (VFI), which is an indicator of retinal perfusion and conventional metrics (such as vessel area density) in the foveal and parafoveal regions. There was a significant difference in the parafoveal VFI in the ictal state between the groups (p = 0.009). During migraine attacks there was a significant reduction in the parafoveal region VFI in MA (-7%, 95% confidence interval [CI] -10% to -4%; p = 0.006) and MO (-7%, 95% CI -10% to -3%; p = 0.016) from their interictal baseline as compared to the change between repeat scans in HCs (2%, 95% CI -3% to 7%). Interictally, there was a mean (standard deviation [SD]) 13% (10%) (p = 0.003) lower blood perfusion in the MA group as compared to the MO group in the foveal region (mean [SD] 0.093 [0.023] vs. 0.107 [0.021], p = 0.003). Interictal analysis also revealed higher circularity in the superficial foveal avascular zone in the MA group compared with the MO group (mean [SD] 0.686 [0.088] vs. 0.629 [0.120], p = 0.004). In addition, interictal analysis of the patients with MA or MO and unilateral headache showed increased retinal vascular parameters consistent with greater perfusion in the eye ipsilateral to the side of the pain as compared with the contralateral eye. These results indicate that perfusion is reduced in MA and MO in the parafoveal retina during the ictal period. Interictally, the foveal retina in MA has reduced perfusion when compared to the foveal retina in MO. Patients with unilateral headache showed interictal asymmetry of retinal perfusion between eyes. These results indicate that changes in retinal perfusion could be a part of migraine pathophysiology, and that distinct retinal vascular signatures identified with OCTA could represent biomarkers for migraine.

STEM-11. A GLIOBLASTOMA SINGLE-CELL META-ATLAS IDENTIFIES NOVEL NEUROVASCULAR PROGENITOR IN TUMORS

Abstract Glioblastomas (GBM) are defined by cell type heterogeneity that underlies treatment resistance and brain invasion. Recent studies have identified reactivation of neurodevelopmental cells and gene expression programs in GBM. Here, we leverage a neurodevelopmental perspective to identify novel gene signatures in GBM. To do so, we assembled a glioblastoma meta-atlas comprised of 73,376 single-cell transcriptomes from 7 published studies. With these data, we developed a novel module generation strategy to mitigate batch effects, enabled analysis of signatures in each tumors, and emphasized modules observed across multiple tumors. Using this approach, we validated known tumor cell types and axes of variation, and also identified novel stemness programs that were uniquely expressed across distinct progenitor populations. Within progenitor subtypes, we identified a neurovascular progenitor (NVP) of cells expressing a mixed vascular signature including genes characteristic of mural and endothelial identity as well as transcriptional programs reminiscent of neural stem cells. Although tumor associated or glioma stem cell derived vasculature has been described as important for tumor formation and progression, the NVP appears to be stem-like. To our knowledge, this population has not been described in GBM. We validated the existence of this NVP with immunofluorescence and fluorescent activated cell sorting from patient tumors followed by single-cell RNA-sequencing. Using a novel organoid transplantation model, we employed single-cell lineage barcoding to identify the progeny of patient-derived NVP cells, determining that they result in various cell types with diverse morphologies. Exploration of data from more traditional model systems including gliomaspheres and mouse GBM models indicate preservation of this NVP identity across systems. We hypothesize that NVP cells may play important roles in tumor plasticity, and thus may be a valuable therapeutic target to explore.

The role of DSC MR perfusion in predicting IDH mutation and 1p19q codeletion status in gliomas: meta-analysis and technical considerations

PurposeIsocitrate dehydrogenase (IDH) mutation and 1p19q codeletion status are important for managing glioma patients. However, current practice dictates invasive tissue sampling for histomolecular classification. We investigated the current value of dynamic susceptibility contrast (DSC) MR perfusion imaging as a tool for the non-invasive identification of these biomarkers.MethodsA systematic search of PubMed, Medline, and Embase up to 2023 was performed, and meta-analyses were conducted. We removed studies employing machine learning models or using multiparametric imaging. We used random-effects standardized mean difference (SMD) and bivariate sensitivity-specificity meta-analyses, calculated the area under the hierarchical summary receiver operating characteristic curve (AUC) and performed meta-regressions using technical acquisition parameters (e.g., time to echo [TE], repetition time [TR]) as moderators to explore sources of heterogeneity. For all estimates, 95% confidence intervals (CIs) are provided.ResultsSixteen eligible manuscripts comprising 1819 patients were included in the quantitative analyses. IDH mutant (IDHm) gliomas had lower rCBV values compared to their wild-type (IDHwt) counterparts. The highest SMD was observed for rCBVmean, rCBVmax, and rCBV 75th percentile (SMD≈ − 0.8, 95% CI ≈ [− 1.2, − 0.5]). In meta-regression, shorter TEs, shorter TRs, and smaller slice thicknesses were linked to higher absolute SMDs. When discriminating IDHm from IDHwt, the highest pooled specificity was observed for rCBVmean (82% [72, 89]), and the highest pooled sensitivity (i.e., 92% [86, 93]) and AUC (i.e., 0.91) for rCBV 10th percentile. In the bivariate meta-regression, shorter TEs and smaller slice gaps were linked to higher pooled sensitivities. In IDHm, 1p19q codeletion was associated with higher rCBVmean (SMD = 0.9 [0.2, 1.5]) and rCBV 90th percentile (SMD = 0.9 [0.1, 1.7]) values.ConclusionsIdentification of vascular signatures predictive of IDH and 1p19q status is a novel promising application of DSC perfusion. Standardization of acquisition protocols and post-processing of DSC perfusion maps are warranted before widespread use in clinical practice.

Cutaneous vascular structure and perfusion in patients with chronic plaque psoriasis.

Vascular dysfunction is a significant contributor to the pathophysiology of psoriasis. Some individuals have variation within the gene for vascular endothelial growth factor-A (VEGF-A), which confers an increased risk of developing psoriasis and having a severe disease phenotype, and may determine responsiveness to treatment. To determine whether patients with psoriasis have alterations in cutaneous microvascular anatomy and physiology due to expression of VEGF and whether laser Doppler imaging has utility in the assessment of this. Twelve adult volunteers with Type 1 chronic plaque psoriasis underwent laser Doppler imaging of plaque and uninvolved skin. Skin biopsies were taken from the areas imaged for immunohistochemistry, including blood and lymphatic vessel markers, and VEGF-A isotype analysis (VEGF-A121, VEGF-A165 and VEGF-D). Venous blood was collected for DNA extraction, VEGF-A genotyping and peripheral blood mononuclear cell culture. Mean blood vessel area (P < 0·01), number of blood vessels (P < 0·001), number of lymphatic vessels (P < 0·001) and blood flow (P < 0·001) was significantly increased in psoriasis plaques, as was expression of VEGF-A121 (P < 0·01), VEGF-A165 (P < 0·04) and VEGF-D (P < 0·01). Blood flow within psoriasis plaques was independent of their increased vascularity (P < 0·01) and may be associated with baseline productivity of VEGF. The number of blood vessels within uninvolved skin in patients with psoriasis was associated with the VEGF-A (rs833061) genotype (P = 0·01), in a relationship suggesting an allele dosing effect. Noninvasive imaging of blood flow may help determine the cutaneous vascular signature for individual patients. This may be a useful prognostic indicator of psoriasis susceptibility and severity, and thus support selection of treatments.

Angiopoietin 2 Is Associated with Vascular Necroptosis Induction in Coronavirus Disease 2019 Acute Respiratory Distress Syndrome

Vascular injury is a well-established, disease-modifying factor in acute respiratory distress syndrome (ARDS) pathogenesis. Recently, coronavirus disease 2019 (COVID-19)–induced injury to the vascular compartment has been linked to complement activation, microvascular thrombosis, and dysregulated immune responses. This study sought to assess whether aberrant vascular activation in this prothrombotic context was associated with the induction of necroptotic vascular cell death. To achieve this, proteomic analysis was performed on blood samples from COVID-19 subjects at distinct time points during ARDS pathogenesis (hospitalized at risk, N = 59; ARDS, N = 31; and recovery, N = 12). Assessment of circulating vascular markers in the at-risk cohort revealed a signature of low vascular protein abundance that tracked with low platelet levels and increased mortality. This signature was replicated in the ARDS cohort and correlated with increased plasma angiopoietin 2 levels. COVID-19 ARDS lung autopsy immunostaining confirmed a link between vascular injury (angiopoietin 2) and platelet-rich microthrombi (CD61) and induction of necrotic cell death [phosphorylated mixed lineage kinase domain-like (pMLKL)]. Among recovery subjects, the vascular signature identified patients with poor functional outcomes. Taken together, this vascular injury signature was associated with low platelet levels and increased mortality and can be used to identify ARDS patients most likely to benefit from vascular targeted therapies.

Binding of Plasmodium falciparum-Infected Red Blood Cells to Engineered 3D Microvessels.

P. falciparum-infected red blood cell (iRBC) sequestration in the microvasculature is a pivotal event in severe malaria pathogenesis. In vitro binding assays using endothelial cell monolayers under static and flow conditions have revealed key ligand-receptor interactions for iRBC sequestration. However, mechanisms remain elusive for iRBC sequestration in specific vascular locations, which prevents further development of effective therapies. New models are needed to better recapitulate the complex geometry of blood flow in human blood vessels and organ-specific vascular signatures. Recent advances in engineering 3D microvessels in vitro have emerged as promising technologies to not only model complex human vascular structures but also allow for precise and step-wise control of individual biological and biomechanical parameters. By designing networks with different branching structures and change of vessel diameter along the flow path, these models recapitulate pressure and flow changes occurring in vivo. Here, we describe the methodology employed to build 3D microvessels using soft lithography and injection molding techniques, as well as the protocol to fabricate capillary-size vessels through collagen photoablation. Furthermore, we describe the methodology of using these models to study malaria and narrate necessary steps for perfusion of P. falciparum through 3D microvessels and different options to quantify P. falciparum-iRBC binding.

Advanced imaging to assess longitudinal vascular changes in brain metastases treated with checkpoint inhibition.

3059 Background: Immune checkpoint inhibitors (ICI) have recently been shown to be effective for brain metastases (BM) in melanoma and lung cancer. Several studies demonstrate that 20-50% of BM patients respond to ICI. The reasons behind this wide variability in treatment response is not clear. Therefore, using physiologic imaging, we seek to identify the longitudinal biological changes exerted on BM as a result of ICI administration. Methods: Given the importance of aberrant tumor vasculature in cancer proliferation, we have focused on assessing changes in vascular physiology. We analyzed standard post-contrast and dynamic susceptibility contrast (DSC) MRI to identify characteristic vascular signatures as part of an ongoing Phase 2 study of pembrolizumab for patients with untreated or progressive, previously treated BM from any histology. Tumor volume measurements were calculated by summating all enhancing voxels. As per modified RECIST and RANO criteria for immunotherapy, volumetric increase of &gt; 40% was defined as progressive disease (PD), a decrease of &gt; 60% as partial response (PR), and stable disease (SD) as between -60% and +40%. Results: 35 patients, out of the total cohort of 60, have undergone DSC-MRI analysis. Histologies include 15 with breast cancer, 6 with non-small cell lung cancer, 4 with melanoma, and 10 with other cancers. At baseline, the total number of BM was 1-50+ per patient. Based on summing the entire enhancing intracranial disease burden, best volumetric responses for the 35 evaluable patients include 4 PR, 12 SD, and 19 PD. Thus far, we found that ICI-resistant BM had a 50% increase in cerebral blood flow (CBF), 105% increase in cerebral blood volume (CBV), a 15% increase in mean transit time (MTT), and an 80% increase in vessel caliber at 6 weeks post-treatment. On the other hand, ICI-responsive BM had no change in CBF, a 33% increase in CBV, a 10% decrease in MTT, and no change in vessel caliber. Ongoing analysis to uncover additional vascular changes (e.g. tumor oxygenation, vessel size index) within BM to ICI are pending. Conclusions: Our data provides evidence that effective ICI for BM is associated with unique intra-tumoral vascular physiology. With final analysis, we will uncover other facets of vascular physiology that correlate with ICI response, and may reveal mechanisms of response/resistance within tumors to ICI.

Neural Processing of Cognitive Control in an Emotionally Neutral Context in Anxiety Patients.

Impaired cognitive control plays a crucial role in anxiety disorders and is associated with deficient neural mechanisms in the fronto-parietal network. Usually, these deficits were found in tasks with an emotional context. The present study aimed at investigating electrophysiological and vascular signatures from event-related brain potentials (ERPs) and functional near-infrared spectroscopy (fNIRS) in anxiety patients versus healthy controls during an inhibition task integrated in an emotionally neutral context. Neural markers were acquired during the completion of a classical Eriksen flanker task. The focus of data analysis has been the ERPs N200 and P300 and fNIRS activations in addition to task performance. No behavioral or neural group differences were identified. ERP findings showed a larger N2pc and a delayed and reduced P300 for incongruent stimuli. The N2pc modulation suggests the reorienting of attention to salient stimuli, while the P300 indicates longer lasting stimulus evaluation processes due to increased task difficulty. FNIRS did not result in any significant activation potentially suggesting a contribution from deeper brain areas not measurable with fNIRS. The missing group difference in our non-emotional task indicates that no generalized cognitive control deficit but rather a more emotionally driven deficit is present in anxiety patients.

Differential effect of vascularity between long- and short-term survivors with IDH1/2 wild-type glioblastoma.

IDH1/2 wt glioblastoma (GB) represents the most lethal tumour of the central nervous system. Tumour vascularity is associated with overall survival (OS), and the clinical relevance of vascular markers, such as rCBV, has already been validated. Nevertheless, molecular and clinical factors may have different influences on the beneficial effect of a favourable vascular signature. To evaluate the association between the rCBV and OS of IDH1/2 wt GB patients for long-term survivors (LTSs) and short-term survivors (STSs). Given that initial high rCBV may affect the patient's OS in follow-up stages, we will assess whether a moderate vascularity is beneficial for OS in both groups of patients. Ninety-nine IDH1/2 wt GB patients were divided into LTSs (OS ≥ 400 days) and STSs (OS < 400 days). Mann-Whitney and Fisher, uni- and multiparametric Cox, Aalen's additive regression and Kaplan-Meier tests were carried out. Tumour vascularity was represented by the mean rCBV of the high angiogenic tumour (HAT) habitat computed through the haemodynamic tissue signature methodology (available on the ONCOhabitats platform). For LTSs, we found a significant association between a moderate value of rCBVmean and higher OS (uni- and multiparametric Cox and Aalen's regression) (p = 0.0140, HR = 1.19; p = 0.0085, HR = 1.22) and significant stratification capability (p = 0.0343). For the STS group, no association between rCBVmean and survival was observed. Moreover, no significant differences (p > 0.05) in gender, age, resection status, chemoradiation, or MGMT methylation were observed between LTSs and STSs. We have found different prognostic and stratification effects of the vascular marker for the LTS and STS groups. We propose the use of rCBVmean at HAT as a vascular marker clinically relevant for LTSs with IDH1/2 wt GB and maybe as a potential target for randomized clinical trials focused on this group of patients.

Dynamic Contrast Enhanced MRI and Intravoxel Incoherent Motion to Identify Molecular Subtypes of Breast Cancer with Different Vascular Normalization Gene Expression.

ObjectiveTo assess the expression of vascular normalization genes in different molecular subtypes of breast cancer and to determine whether molecular subtypes with a higher vascular normalization gene expression can be identified using dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) and intravoxel incoherent motion (IVIM) diffusion-weighted imaging (DWI).Materials and MethodsThis prospective study evaluated 306 female (mean age ± standard deviation, 50 ± 10 years), recruited between January 2014 and August 2017, who had de novo breast cancer larger than 1 cm in diameter (308 tumors). DCE MRI followed by IVIM DWI studies using 11 different b-values (0 to 1200 s/mm2) were performed on a 1.5T MRI system. The Tofts model and segmented biexponential IVIM analysis were used. For each tumor, the molecular subtype (according to six [I-VI] subtypes and PAM50 subtypes), expression profile of genes for vascular normalization, pericytes, and normal vascular signatures were determined using freshly frozen tissue. Statistical associations between imaging parameters and molecular subtypes were examined using logistic regression or linear regression with a significance level of p = 0.05.ResultsBreast cancer subtypes III and VI and PAM50 subtypes luminal A and normal-like exhibited a higher expression of genes for vascular normalization, pericyte markers, and normal vessel function signature (p < 0.001 for all) compared to other subtypes. Subtypes III and VI and PAM50 subtypes luminal A and normal-like, versus the remaining subtypes, showed significant associations with Ktrans, kep, vp, and IAUGCBN90 on DEC MRI, with relatively smaller values in the former. The subtype grouping was significantly associated with D, with relatively less restricted diffusion in subtypes III and VI and PAM50 subtypes luminal A and normal-like.ConclusionDCE MRI and IVIM parameters may identify molecular subtypes of breast cancers with a different vascular normalization gene expression.

Undescribed Vascular Signatures: A Contraindication to Ultrasound-guided Parasagittal Infraclavicular Block! A Retrospective Observational Study.

Background:The ultrasound-infraclavicular block (US-ICB) is a popular and efficient block for below-elbow surgeries. However, the vascular anatomy of infraclavicular area close to the brachial plexus has remained unresearched. We aimed to explore the presence of aberrant vasculature in the infraclavicular area that could pose a contraindication to US-ICB.Methods:In this retrospective observational study, we reviewed the US images of patients undergoing below-elbow surgery under US-ICB. Before performing the block, a scout scan of parasagittal infraclavicular areas was performed and the scan images were saved. The primary objective was to find the prevalence of aberrant vasculature due to which the US–ICB was abandoned. The secondary objective was to understand the pattern and position of the aberrant vessels.Results:Out of 912 patients, 793 patients underwent surgery under US–ICB and in 119 patients (13.05%), the USG-ICB was abandoned due to aberrant vasculature close to the brachial cords and intended position of the needle tip. The anomalous vessels were identified in the lower inner, lower outer, and upper outer quadrants around the axillary artery (AA). Some of these vascular structures also had classical patterns which we described as “satellites,” “clamping,” or “hugging” of the AA.Conclusion:Anomalous vascular structures in the infraclavicular area were seen in 13.05% of patients planned for US–ICB. We, therefore, recommend, that a thorough scout US scan should be mandatorily performed ICB and in the presence of aberrant vascular structures, an alternative approach to brachial plexus block may be adopted.

NIMG-05. ADVANCED IMAGING TO ASSESS LONGITUDINAL VASCULAR CHANGES IN BRAIN METASTASES TREATED WITH CHECKPOINT INHIBITION

Abstract Immune checkpoint inhibitors (ICI) have recently been shown to be effective for brain metastases (BM) in melanoma and lung cancer. However, accurately assessing intracranial response in patients undergoing ICI is a challenge, as current measures cannot reliably distinguish pseudoprogression from true tumor progression. To identify potential biomarkers of response, we analyzed standard post-contrast and dynamic susceptibility contrast MRI to identify characteristic vascular signatures as part of an ongoing Phase 2 study of pembrolizumab for patients with untreated or progressive, previously treated BM from any histology. Tumor volume measurements were calculated by summating all enhancing voxels. A volumetric increase of &amp;gt;40% was categorized as progressive disease (PD), a decrease of &amp;gt;60% as partial response (PR), and stable disease (SD) as between -60% and +40%. 78 patients have been enrolled, of whom 44 have received at least baseline advanced MR imaging. Histologies include 21 with breast cancer, 5 with non-small cell lung cancer, 4 with melanoma, and 13 with other cancers. At baseline, the total number of BM was 1-50+ per patient. Based on summing the entire enhancing intracranial disease burden, best volumetric responses for the 33 evaluable patients include 4 PR, 10 SD, and 19 PD. On preliminary analysis, there was a correlation between increased tumor cerebral blood volume/flow with tumor progression. Correlation of additional vascular physiologic parameters (e.g. vessel caliber, tissue oxygenation) to volumetric response, patient outcome, and standardized response criteria (iRANO) are ongoing. Our data provides potential evidence that effective ICI is associated with a decrease in perfusion. Ongoing analyses to uncover additional vascular changes – specifically longitudinal metrics reflecting vascular structure and function - within BM to ICI are pending. These findings have potential to explore mechanisms of ICI response and resistance, as well as biomarkers of response.

Advanced imaging to assess longitudinal vascular changes in brain metastases treated with immune checkpoint inhibition.

2529 Background: Immune checkpoint inhibitors (ICI) have recently been shown to be effective for brain metastases (BM) for melanoma and lung cancer. This breakthrough has prompted interest in evaluating ICI in BM of other histologies. However, accurately assessing intracranial response in patients undergoing ICI is a challenge, as current measures cannot distinguish pseudoprogression from true tumor progression. To shed light on potential biomarkers of response, we prospectively use perfusion MRI to identify characteristic vascular signatures in a BM-specific trial of ICI. Methods: As part of an ongoing phase II study of pembrolizumab for patients with untreated or progressive, previously treated BM from any histology, patients underwent advanced MRI that includes tumor volume measurements and perfusion imaging with dynamic susceptibility contrast MRI. To calculate volumetric radiographic response, all enhancing voxels were summated. A volumetric increase of &gt;40% was categorized as progressive disease (PD), a decrease of &gt;60% as partial response (PR), and stable disease (SD) as between -60% and +40%. Results: 53 patients have been enrolled, of whom 44 have received at least baseline advanced MR imaging. Histologies include 21 with breast cancer, 5 with non-small cell lung cancer, 4 with melanoma, and 13 with other cancers. At baseline, the total number of BM was 1-50+ per patient. Based on summing the entire enhancing intracranial disease burden, best volumetric responses for the 33 evaluable patients include 4 PR, 10 SD, and 19 PD. On preliminary analysis, there was a correlation between increased tumor cerebral blood volume/flow with tumor progression. Correlation of additional vascular physiologic parameters (e.g. vessel caliber, tissue oxygenation) and volumetric response to patient outcome and standardized response criteria (iRANO) are ongoing. Conclusions: Pembrolizumab likely has anti-tumor efficacy in BM. Our data provides potential evidence that effective ICI is associated with a decrease in perfusion. Ongoing analyses to uncover additional vascular changes – specifically longitudinal metrics reflecting vascular structure and function - within BM to ICI are pending. These findings have potential to illustrate mechanisms of efficacy for ICI and biomarkers of response in this patient population.

Vascular dysfunction promotes regional hypoxia after bevacizumab therapy in recurrent glioblastoma patients

BackgroundHypoxia is a driver of treatment resistance in glioblastoma. Antiangiogenic agents may transiently normalize blood vessels and decrease hypoxia before excessive pruning of vessels increases hypoxia. The time window of normalization is dose and time dependent. We sought to determine how VEGF blockade with bevacizumab modulates tumor vasculature and the impact that those vascular changes have on hypoxia in recurrent glioblastoma patients.MethodsWe measured tumor volume, vascular permeability (Ktrans), perfusion parameters (cerebral blood flow/volume, vessel caliber, and mean transit time), and regions of hypoxia in patients with recurrent glioblastoma before and after treatment with bevacizumab alone or with lomustine using [18F]FMISO PET-MRI. We also examined serial changes in plasma biomarkers of angiogenesis and inflammation.ResultsEleven patients were studied. The magnitude of global tumor hypoxia was variable across these 11 patients prior to treatment and it did not significantly change after bevacizumab. The hypoxic regions had an inefficient vasculature characterized by elevated cerebral blood flow/volume and increased vessel caliber. In a subset of patients, there were tumor subregions with decreased mean transit times and a decrease in hypoxia, suggesting heterogeneous improvement in vascular efficiency. Bevacizumab significantly changed known pharmacodynamic biomarkers such as plasma VEGF and PlGF.ConclusionsThe vascular signature in hypoxic tumor regions indicates a disorganized vasculature which, in most tumors, does not significantly change after bevacizumab treatment. While some tumor regions showed improved vascular efficiency following treatment, bevacizumab did not globally alter hypoxia or normalize tumor vasculature in glioblastoma.

Molecular determinants of nephron vascular specialization in the kidney

Although kidney parenchymal tissue can be generated in vitro, reconstructing the complex vasculature of the kidney remains a daunting task. The molecular pathways that specify and sustain functional, phenotypic and structural heterogeneity of the kidney vasculature are unknown. Here, we employ high-throughput bulk and single-cell RNA sequencing of the non-lymphatic endothelial cells (ECs) of the kidney to identify the molecular pathways that dictate vascular zonation from embryos to adulthood. We show that the kidney manifests vascular-specific signatures expressing defined transcription factors, ion channels, solute transporters, and angiocrine factors choreographing kidney functions. Notably, the ontology of the glomerulus coincides with induction of unique transcription factors, including Tbx3, Gata5, Prdm1, and Pbx1. Deletion of Tbx3 in ECs results in glomerular hypoplasia, microaneurysms and regressed fenestrations leading to fibrosis in subsets of glomeruli. Deciphering the molecular determinants of kidney vascular signatures lays the foundation for rebuilding nephrons and uncovering the pathogenesis of kidney disorders.

A Genome-wide Analysis of Human Pluripotent Stem Cell-Derived Endothelial Cells in 2D or 3D Culture

SummaryA defined protocol for efficiently deriving endothelial cells from human pluripotent stem cells was established and vascular morphogenesis was used as a model system to understand how synthetic hydrogels influence global biological function compared with common 2D and 3D culture platforms. RNA sequencing demonstrated that gene expression profiles were similar for endothelial cells and pericytes cocultured in polyethylene glycol (PEG) hydrogels or Matrigel, while monoculture comparisons identified distinct vascular signatures for each cell type. Endothelial cells cultured on tissue-culture polystyrene adopted a proliferative phenotype compared with cells cultured on or encapsulated in PEG hydrogels. The proliferative phenotype correlated to increased FAK-ERK activity, and knockdown or inhibition of ERK signaling reduced proliferation and expression for cell-cycle genes while increasing expression for “3D-like” vasculature development genes. Our results provide insight into the influence of 2D and 3D culture formats on global biological processes that regulate cell function.

Arterial Stiffness, Thickness and Association to Suitable Novel Markers of Risk at the Origin of Cardiovascular Disease in Obese Children.

Atherosclerosis origins early in childhood. Aim of the study was to investigate vascular signature and phenotypes of cardiovascular disease in obese children and adolescents identifying novel potential circulating markers of risk.Cross-sectional study of intima-media-thickness (IMT), pulse wave velocity (PWV), augmentation index (AIX@75), circulating markers (E-selectin, soluble-intercellular-adhesion-molecule1_ICAM1, chemerin, fatty-acid-binding protein 4, sCD36, lipopolysaccharides_LPS, oxLDL, fetuin) in 123 obese (body mass index, BMI z-score >1.645 SD) children (N=55, age ≤10 years-old) and adolescents (N=68, age >10 years-old).Adolescents had significantly higher uric acid (p=0.002), non-HDL-cholesterol (p=0.02), fasting glucose (p=0.04), systolic blood pressure (p=0.005) and PWV (p=0.02) than children.Obese adolescent patients with metabolic syndrome (MetS) abnormalities had higher PWV (p<0.05) than peers without. No differences were observed in circulating biomarkers in relationship to age and MetS status. oxLDL, sCD36 and LPS were correlated to AIX@75 and/or IMTM in children and adolescents (p ranging from <0.05 to <0.0001). Total cholesterol, non-HDL-cholesterol, TG/HDL ratio, oxLDL, sCD36, ICAM1, LPS were significantly different across AIX@75 tertiles (p between 0.03 and 0.001).Early phenotypes of cardiovascular alterations in young severely obese patients encompass increased IMT, stiffness of intermediate size and resistance vasculature. Novel biomarkers investigated in the present study were associated to estimates of stiffness and thickness not differently from traditional risk factor such as non-HDL-cholesterol and TG/HDL ratio.

Acute Temporal Changes of MRI-Tracked Tumor Vascular Parameters after Combined Anti-angiogenic and Radiation Treatments in a Rat Glioma Model: Identifying Signatures of Synergism.

In this study we used magnetic resonance imaging (MRI) biomarkers to monitor the acute temporal changes in tumor vascular physiology with the aim of identifying the vascular signatures that predict response to combined anti-angiogenic and radiation treatments. Forty-three athymic rats implanted with orthotopic U-251 glioma cells were studied for approximately 21 days after implantation. Two MRI studies were performed on each animal, pre- and post-treatment, to measure tumor vascular parameters. Two animal groups received treatment comprised of Cilengitide, an anti-angiogenic agent and radiation. The first group received a subcurative regimen of Cilengitide 1 h before irradiation, while the second group received a curative regimen of Cilengitide 8 h before irradiation. Cilengitide was given as a single dose (4 mg/kg; intraperitoneal) after the pretreatment MRI study and before receiving a 20 Gy radiation dose. After irradiation, the post-treatment MRI study was performed at selected time points: 2, 4, 8 and 12 h (n = ≥5 per time point). Significant changes in vascular parameters were observed at early time points after combined treatments in both treatment groups (1 and 8 h). The temporal changes in vascular parameters in the first group (treated 1 h before exposure) resembled a previously reported pattern associated with radiation exposure alone. Conversely, in the second group (treated 8 h before exposure), all vascular parameters showed an initial response at 2-4 h postirradiation, followed by an apparent lack of response at later time points. The signature time point to define the "synergy" of Cilengitide and radiation was 4 h postirradiation. For example, 4 h after combined treatments using a 1 h separation (which followed the subcurative regimen), tumor blood flow was significantly decreased, nearly 50% below baseline (P = 0.007), whereas 4 h after combined treatments using an 8 h separation (which followed the curative regimen), tumor blood flow was only 10% less than baseline. Comparison between the first and second groups further revealed that most other vascular parameters were maximally different 4 h after combined treatments. In conclusion, the data are consistent with the assertion that the delivery of radiation at the vascular normalization time window of Cilengitide improves radiation treatment outcome. The different vascular responses after the different delivery times of combined treatments in light of the known tumor responses under similar conditions would indicate that timing has a crucial influence on treatment outcome and long-term survival. Tracking acute changes in tumor physiology after monotherapy or combined treatments appears to aid in identifying the beneficial timing for administration, and perhaps has predictive value. Therefore, judicial timing of treatments may result in optimal treatment response.

Direct Conversion of Adult Endothelial Cells into Immunecompetent Long-Term Engraftable Clinically Scalable Hematopoietic Stem Cells: Pathway to Therapeutic Translation

▪The molecular pathways and microenvironmental cues that choreograph the conversion of endothelial cells (ECs) into true engraftable hematopoietic stem cells (HSCs) remain undefined. This is due to lack of models to recreate the short-lived transition from EC to hemogenic cells and to HSCs. Extending on our previous work (Sandler V. et al, Reprogramming of human endothelium into hematopoietic cells requires vascular niche induction. Nature, 511:312-8. 2014), we have developed a novel, sequential, clinically-translatable in vitro model of the adult EC to hematopoietic transition (EHT). This model uses precise, conditional, on-off expression of transcription factors (FosB, Gfi1, Runx1, and Spi1 - FGRS) and an inductive vascular niche to reprogram adult mouse ECs into true HSCs (rEC-HSCs) with high efficiency. During the induction phase (days 0-8), FGRS are conditionally expressed in adult non-lymphatic ECs isolated from Runx1-IRES-GFP reporter mice and co-cultured with the supportive vascular niche cells. During the specification phase (days 8-20), FGRS-transduced VEcad+Runx1-CD45- ECs activate expression of endogenous Runx1, initiating the hematopoietic program and silencing EC fate. The VEcad+Runx1+CD45+ cells, then complete specification and full commitment to VEcad-Runx1+CD45+ hematopoietic stem and progenitor cells (rEC-HSPCs). Specified rEC-HSPCs are then expanded (days 20-28) on the vascular niche generating a large number of hematopoietic cells and, at this point, expression of exogenous FGRS is turned off. Transplantation of rEC-HSPCs (CD45.2) into lethally irradiated (CD45.1) recipient mice reconstitute both short-term and long-term hematopoiesis, and are capable of engrafting secondary and tertiary recipients (rEC-HSCs). Once engrafted, rEC-HSPCs give rise to functional myeloid and lymphoid cells with full complement of polarized T cell subsets. rEC-HSC-derived immune cells undergo T-cell receptor (TCR) rearrangement and reconstitute adaptive immune function in Rag1-/- mice.To prove the stem cell potential of rEC-HSCs, we performed clonal analyses on the VEcad+Runx1+CD45+ cells (days 8-20), in which single cells were plated in coculture with vascular niche. Notably, 7 out of 386 CD45.2+ clones gave rise to expanding colonies that were capable of 4 months primary multilineage engraftment into lethally irradiated CD45.1+ recipients. In addition, limiting dilution transplantation of isochronic VEcad-Runx1+CD45+ cells indicated that 1 out of 538 rEC-HSPCs are repopulating rEC-HSCs with primary and secondary engraftment potential. Thus, based on both clonal and limiting dilution transplantation studies, we demonstrate that rEC-HSCs represent true repopulating prototypical HSCs. In addition, since during the expansion phase there are >200 fold expansion of the SLAM-coded KLS population, these data support the notion that during the 20-28 days expansion phase there is robust proliferation of rEC-HSCs. Serial analyses of the primary, secondary and tertiary engrafted rEC-HSCs, showed no evidence of leukemias. Molecular analyses indicated that both in vitro and in vivo expanded rEC-HSCs had complete erasure of vascular signature and stable expression of hematopoietic profile.Moreover, employing Runx1-IRES-GFP reporter mice enabled deconvolution of stage-specific pathways involved in generation of engraftable rEC-HSCs. Inhibition of TGFβ signaling along with activation of BMP and CXCL12 pathways reinforced the induction phase. Active Notch and CXCL12 signaling throughout the specification and expansion phases supported self-renewal of transplantable rEC-HSCs. This stepwise reprogramming approach provides an interrogatable in vitro platform to elucidate the critical pathways involved in the transition of ECs into hemogenic-like and ultimately hematopoietic cells in vitro. Specifically, these data unequivocally support that our approach lead to generation of large number HSCs, enabling therapeutic application. Thus, our reprogramming platform that does not require transition through a pluripotent state, will facilitate devising strategies to reprogram ECs into abundant autologous long-term repopulating HSCs amenable to genetic modification for treatment of inherited and acquired hematological disorders. DisclosuresGinsberg:Angiocrine Bioscience: Employment. Butler:Angiocrine Bioscience: Research Funding. Rafii:Angiocrine Bioscience: Equity Ownership, Other: Non-paid consultant.

PML-RARa modulates the vascular signature of extracellular vesicles released by acute promyelocytic leukemia cells.

Oncogenic transformation is believed to impact the vascular phenotype and microenvironment in cancer, at least in part, through mechanisms involving extracellular vesicles (EVs). We explored these questions in the context of acute promyelocytic leukemia cells (NB4) expressing oncogenic fusion protein, PML-RARa and exquisitely sensitive to its clinically used antagonist, the all-trans retinoic acid (ATRA). We report that NB4 cells produce considerable numbers of EVs, which are readily taken up by cultured endothelial cells triggering their increased survival. NB4 EVs contain PML-RARa transcript, but no detectable protein, which is also absent in endothelial cells upon the vesicle uptake, thereby precluding an active intercellular trafficking of this oncogene in this setting. ATRA treatment changes the emission profile of NB4-related EVs resulting in preponderance of smaller vesicles, an effect that occurs in parallel with the onset of cellular differentiation. ATRA also increases IL-8 mRNA and protein content in NB4 cells and their EVs, while decreasing the levels of VEGF and tissue factor (TF). Endothelial cell uptake of NB4-derived EVs renders these cells more TF-positive and procoagulant, and this effect is diminished by pre-treatment of EV donor cells with ATRA. Profiling angiogenesis-related transcripts in intact and ATRA-treated APL cells and their EVs reveals multiple differences attributable to cellular responses and EV molecular packaging. These observations point to the potential significance of changes in the angiogenic signature and activity associated with EVs released from tumor cells subjected to targeted therapy.