Vessel Maturation Research Articles

3D-bioprinted gradient-structured scaffold generates anisotropic cartilage with vascularization by pore-size-dependent activation of HIF1α/FAK signaling axis

Articular cartilage injury is one of the most common diseases in orthopedics, which seriously affects patients’ life quality, the development of a biomimetic scaffold that mimics the multi-layered gradient structure of native cartilage is a new cartilage repair strategy. It has been shown that scaffold topography affects cell attachment, proliferation, and differentiation; the underlying molecular mechanism of cell-scaffold interaction is still unclear. In the present study, we construct an anisotropic gradient-structured cartilage scaffold by three-dimensional (3D) bioprinting, in which bone marrow stromal cell (BMSC)-laden anisotropic hydrogels micropatterns were used for heterogeneous chondrogenic differentiation and physically gradient synthetic poly (ε-caprolactone) (PCL) to impart mechanical strength. In vitro and in vivo, we demonstrated that gradient-structured cartilage scaffold displayed better cartilage repair effect. The heterogeneous cartilage tissue maturation and blood vessel ingrowth were mediated by a pore-size-dependent mechanism and HIF1α/FAK axis activation. In summary, our results provided a theoretical basis for employing 3D bioprinting gradient-structured constructs for anisotropic cartilage regeneration and revealed HIF1α/FAK axis as a crucial regulator for cell-material interactions, so as to provide a new perspective for cartilage regeneration and repair.

Hypoxic–ischemic injury causes functional and structural neurovascular degeneration in the juvenile mouse retina

Ischemic stroke is a major cause of long-term disabilities, including vision loss. Neuronal and blood vessel maturation can affect the susceptibility of and outcome after ischemic stroke. Although we recently reported that exposure of neonatal mice to hypoxia–ischemia (HI) severely compromises the integrity of the retinal neurovasculature, it is not known whether juvenile mice are similarly impacted. Here we examined the effect of HI injury in juvenile mice on retinal structure and function, in particular the susceptibility of retinal neurons and blood vessels to HI damage. Our studies demonstrated that the retina suffered from functional and structural injuries, including reduced b-wave, thinning of the inner retinal layers, macroglial remodeling, and deterioration of the vasculature. The degeneration of the retinal vasculature associated with HI resulted in a significant decrease in the numbers of pericytes and endothelial cells as well as an increase in capillary loss. Taken together, these findings suggest a need for juveniles suffering from ischemic stroke to be monitored for changes in retinal functional and structural integrity. Thus, there is an emergent need for developing therapeutic approaches to prevent and reverse retinal neurovascular dysfunction with exposure to ischemic stroke.

Smooth Muscle Specific Ablation of CXCL12 in Mice Downregulates CXCR7 Associated with Defective Coronary Arteries and Cardiac Hypertrophy.

The chemokine CXCL12 plays a fundamental role in cardiovascular development, cell trafficking, and myocardial repair. Human genome-wide association studies even have identified novel loci downstream of the CXCL12 gene locus associated with coronary artery disease and myocardial infarction. Nevertheless, cell and tissue specific effects of CXCL12 are barely understood. Since we detected high expression of CXCL12 in smooth muscle (SM) cells, we generated a SM22-alpha-Cre driven mouse model to ablate CXCL12 (SM-CXCL12−/−). SM-CXCL12−/− mice revealed high embryonic lethality (50%) with developmental defects, including aberrant topology of coronary arteries. Postnatally, SM-CXCL12−/− mice developed severe cardiac hypertrophy associated with fibrosis, apoptotic cell death, impaired heart function, and severe coronary vascular defects characterized by thinned and dilated arteries. Transcriptome analyses showed specific upregulation of pathways associated with hypertrophic cardiomyopathy, collagen protein network, heart-related proteoglycans, and downregulation of the M2 macrophage modulators. CXCL12 mutants showed endothelial downregulation of the CXCL12 co-receptor CXCR7. Treatment of SM-CXCL12−/− mice with the CXCR7 agonist TC14012 attenuated cardiac hypertrophy associated with increased pERK signaling. Our data suggest a critical role of smooth muscle-specific CXCL12 in arterial development, vessel maturation, and cardiac hypertrophy. Pharmacological stimulation of CXCR7 might be a promising target to attenuate adverse hypertrophic remodeling.

FC 102PD INDUCED ARTERIOLAR AND PERITONEAL PATHOMECHANISMS ARE PARTIALLY REVERSED AFTER KIDNEY TRANSPLANTATION

Abstract Background and Aims Due to the unphysiological composition of PD fluids, chronic peritoneal dialysis (PD) induces progressive peritoneal fibrosis, hypervascularization, and vasculopathy. The evolution of the PD membrane and vasculopathy following kidney transplantation (KTx) is largely unknown. Method Arteriolar and peritoneal tissues were obtained from 107 children with chronic kidney disease (CKD5), 72 children on PD (treated with neutral pH PD fluids, with low glucose degradation product content, GDP) and 21 children, who underwent KTx 4-5 weeks after a median 21 months of PD. Specimen underwent standardized digital quantitative histomorphometry. Molecular mechanisms were studied in omental arterioles microdissected from surrounding fat by multi-omics followed by Gene Set Enrichment Analysis (GSEA); key findings were validated in parietal tissues of independent, matched cohorts by quantitative immunohistochemistry (n=15/group). Results Arteriolar transcriptome and proteome GSEA revealed suppression of leucocyte migration and T-cell activation / secretory pathways regulation, of sprouting angiogenesis biological processes and of epithelial proliferation and cell cycle after KTx as compared to PD. Lipid / fatty acid metabolism, autophagy and ATP synthesis pathways were activated. Transcriptome analysis including KTx, PD and CKD5 specifically attributed regulation of arteriolar lipid and fatty acid metabolism to transplantation and comprised 140 transcripts; their regulation was confirmed on the proteome level. Hub gene fatty acid synthase was identified by protein interaction analysis (string-db.org). 15 arteriolar genes activated by PD were inactivated after KTx and included glucose metabolisms and cytoskeleton related transcripts. 24 transcripts and 10 corresponding proteins induced by PD were still active after KTx and associated with biological processes related to TGF-ß signaling, fibrosis and mineral absorption. In line with arteriolar multi-omics findings, peritoneal hypervascularization induced by chronic PD was reversed after Tx to CKD5 level. CD45 positive tissue infiltrating leucocytes count was reduced by 40% and was independently associated with microvessel density in multivariable analysis including PD vintage, daily GDP exposure and recent KTx. Peritoneal lymphatic vessel density, submesothelial thickness, activated fibroblast, fibrin deposit, macrophage and EMT cell counts remained unchanged after KTx compared to PD. Arteriolar lumen to vessel ratios (a marker of vasculopathy) were similar in both groups. Vessel-homeostasis-related proteins in independent, matched cohorts demonstrated increased caspase-3 abundance in peritoneal arterioles after KTx. Arteriolar VEGF-A, thrombospondin, angiopoietin1/2, and hypoxia-inducible factor-1 (HIF-1a) were unchanged, while submesothelial HIF-1a and angiopoietin1/2 were decreased after Tx, favoring vessel maturation. The abundance of the key driver of fibrosis, TGF-ß-effector pSMAD2/3, was unchanged in the peritoneum and arterioles after Tx. Conclusion Our multi-omics analyses of fat covered omental arterioles, not directly exposed to PD fluids, demonstrate inhibition of PD induced immune response and angiogenesis pathways, of glucose metabolism and cytoskeleton regulation to levels similar as seen in children with CKD5. Arteriolar lipid and fatty acid metabolism is selectively altered after KTx. Reversal of low GDP PD induced hypervascularization and inflammation of the parietal peritoneum after KTx, mirror molecular changes in omental arterioles, while profibrotic activity persists after KTx in omental arterioles and in the parietal peritoneum.

The Effect of Inflammatory Priming on the Therapeutic Potential of Mesenchymal Stromal Cells for Spinal Cord Repair

Mesenchymal stromal cells (MSC) are used for cell therapy for spinal cord injury (SCI) because of their ability to support tissue repair by paracrine signaling. Preclinical and clinical research testing MSC transplants for SCI have revealed limited success, which warrants the exploration of strategies to improve their therapeutic efficacy. MSC are sensitive to the microenvironment and their secretome can be altered in vitro by exposure to different culture media. Priming MSC with inflammatory stimuli increases the expression and secretion of reparative molecules. We studied the effect of macrophage-derived inflammation priming on MSC transplants and of primed MSC (pMSC) acute transplants (3 days) on spinal cord repair using an adult rat model of moderate–severe contusive SCI. We found a decrease in long-term survival of pMSC transplants compared with unprimed MSC transplants. With a pMSC transplant, we found significantly more anti-inflammatory macrophages in the contusion at 4 weeks post transplantation (wpt). Blood vessel presence and maturation in the contusion at 1 wpt was similar in rats that received pMSC or untreated MSC. Nervous tissue sparing and functional recovery were similar across groups. Our results indicate that macrophage-derived inflammation priming does not increase the overall therapeutic potential of an MSC transplant in the adult rat contused spinal cord.

DPSCs treated by TGF-\u03b21 regulate angiogenic sprouting of three-dimensionally co-cultured HUVECs and DPSCs through VEGF-Ang-Tie2 signaling

BackgroundMaintaining the stability and maturation of blood vessels is of paramount importance for the vessels to carry out their physiological function. Smooth muscle cells (SMCs), pericytes, and mesenchymal stem cells (MSCs) are involved in the maturation process of the newly formed vessels. The aim of this study was to investigate whether transforming growth factor beta 1 (TGF-β1) treatment could enhance pericyte-like properties of dental pulp stem cells (DPSCs) and how TGF-β1-treated DPSCs for 7 days (T-DPSCs) stabilize the newly formed blood vessels.MethodsWe utilized TGF-β1 to treat DPSCs for 1, 3, 5, and 7 days. Western blotting and immunofluorescence were used to analyze the expression of SMC markers. Functional contraction assay was conducted to assess the contractility of T-DPSCs. The effects of T-DPSC-conditioned media (T-DPSC-CM) on human umbilical vein endothelial cell (HUVEC) proliferation and migration were examined by MTT, wound healing, and trans-well migration assay. Most importantly, in vitro 3D co-culture spheroidal sprouting assay was used to investigate the regulating role of vascular endothelial growth factor (VEGF)-angiopoietin (Ang)-Tie2 signaling on angiogenic sprouting in 3D co-cultured spheroids of HUVECs and T-DPSCs. Angiopoietin 2 (Ang2) and VEGF were used to treat the co-cultured spheroids to explore their roles in angiogenic sprouting. Inhibitors for Tie2 and VEGFR2 were used to block Ang1/Tie2 and VFGF/VEGFR2 signaling.ResultsWestern blotting and immunofluorescence showed that the expression of SMC-specific markers (α-SMA and SM22α) were significantly increased after treatment with TGF-β1. Contractility of T-DPSCs was greater compared with that of DPSCs. T-DPSC-CM inhibited HUVEC migration. In vitro sprouting assay demonstrated that T-DPSCs enclosed HUVECs, resembling pericyte-like cells. Compared to co-culture with DPSCs, a smaller number of HUVEC sprouting was observed when co-cultured with T-DPSCs. VEGF and Ang2 co-stimulation significantly enhanced sprouting in HUVEC and T-DPSC co-culture spheroids, whereas VEGF or Ang2 alone exerted insignificant effects on HUVEC sprouting. Blocking Tie2 signaling reversed the sprouting inhibition by T-DPSCs, while blocking VEGF receptor (VEGFR) signaling boosted the sprouting inhibition by T-DPSCs.ConclusionsThis study revealed that TGF-β1 can induce DPSC differentiation into functional pericyte-like cells. T-DPSCs maintain vessel stability through Ang1/Tie2 and VEGF/VEGFR2 signaling.

Notch regulates vascular collagen IV basement membrane through modulation of lysyl hydroxylase 3 trafficking

Collagen type IV (Col IV) is a basement membrane protein associated with early blood vessel morphogenesis and is essential for blood vessel stability. Defects in vascular Col IV deposition are the basis of heritable disorders, such as small vessel disease, marked by cerebral hemorrhage and drastically shorten lifespan. To date, little is known about how endothelial cells regulate the intracellular transport and selective secretion of Col IV in response to angiogenic cues, leaving a void in our understanding of this critical process. Our aim was to identify trafficking pathways that regulate Col IV deposition during angiogenic blood vessel development. We have identified the GTPase Rab10 as a major regulator of Col IV vesicular trafficking during vascular development using both in vitro imaging and biochemistry as well as in vivo models. Knockdown of Rab10 reduced de novo Col IV secretion in vivo and in vitro. Mechanistically, we determined that Rab10 is an indirect mediator of Col IV secretion, partnering with atypical Rab25 to deliver the enzyme lysyl hydroxylase 3 (LH3) to Col IV-containing vesicles staged for secretion. Loss of Rab10 or Rab25 results in depletion of LH3 from Col IV-containing vesicles and rapid lysosomal degradation of Col IV. Furthermore, we demonstrate that Rab10 is Notch responsive, indicating a novel connection between permissive Notch-based vessel maturation programs and vesicle trafficking. Our results illustrate both a new trafficking-based component in the regulated secretion of Col IV and how this vesicle trafficking program interfaces with Notch signaling to fine-tune basement membrane secretion during blood vessel development.

Long-Term Imaging of Wound Angiogenesis with Large Scale Optoacoustic Microscopy.

Wound healing is a well‐coordinated process, necessitating efficient formation of new blood vessels. Vascularization defects are therefore a major risk factor for chronic, non‐healing wounds. The dynamics of mammalian tissue revascularization, vessel maturation, and remodeling remain poorly understood due to lack of suitable in vivo imaging tools. A label‐free large‐scale optoacoustic microscopy (LSOM) approach is developed for rapid, non‐invasive, volumetric imaging of tissue regeneration over large areas spanning up to 50 mm with a depth penetration of 1.5 mm. Vascular networks in dorsal mouse skin and full‐thickness excisional wounds are imaged with capillary resolution during the course of healing, revealing previously undocumented views of the angiogenesis process in an unperturbed wound environment. Development of an automatic analysis framework enables the identification of key features of wound angiogenesis, including vessel length, diameter, tortuosity, and angular alignment. The approach offers a versatile tool for preclinical research in tissue engineering and regenerative medicine, empowering label‐free, longitudinal, high‐throughput, and quantitative studies of the microcirculation in processes associated with normal and impaired vascular remodeling, and analysis of vascular responses to pharmacological interventions in vivo.

Different Forms of Tumor Vascularization and Their Clinical Implications Focusing on Vessel Co-option in Colorectal Cancer Liver Metastases.

In modern anti-cancer therapy of metastatic colorectal cancer (mCRC) the anti-angiogenic treatment targeting sprouting angiogenesis is firmly established for more than a decade. However, its clinical benefits still remain limited. As liver metastases (LM) represent the most common metastatic site of colorectal cancer and affect approximately one-quarter of the patients diagnosed with this malignancy, its treatment is an essential aspect for patients' prognosis. Especially in the perioperative setting, the application of anti-angiogenic drugs represents a therapeutic option that may be used in case of high-risk or borderline resectable colorectal cancer liver metastases (CRCLM) in order to achieve secondary resectability. Regarding CRCLM, one reason for the limitations of anti-angiogenic treatment may be represented by vessel co-option (VCO), which is an alternative mechanism of blood supply that differs fundamentally from the well-known sprouting angiogenesis and occurs in a significant fraction of CRCLM. In this scenario, tumor cells hijack pre-existing mature vessels of the host organ independently from stimulating new vessels formation. This represents an escape mechanism from common anti-angiogenic anti-cancer treatments, as they primarily target the main trigger of sprouting angiogenesis, the vascular endothelial growth factor A. Moreover, the mechanism of blood supply in CRCLM can be deduced from their phenotypic histopathological growth pattern (HGP). For that, a specific guideline has already been implemented. These HGP vary not only regarding their blood supply, but also concerning their tumor microenvironment (TME), as notable differences in immune cell infiltration and desmoplastic reaction surrounding the CRCLM can be observed. The latter actually serves as one of the central criteria for the classification of the HGP. Regarding the clinically relevant effects of the HGP, it is still a topic of research whether the VCO-subgroup of CRCLM results in an impaired treatment response to anti-angiogenic treatment when compared to an angiogenic subgroup. However, it is well-proved, that VCO in CRCLM generally relates to an inferior survival compared to the angiogenic subgroup. Altogether the different types of blood supply result in a relevant influence on the patients' prognosis. This reinforces the need of an extended understanding of the underlying mechanisms of VCO in CRCLM with the aim to generate more comprehensive approaches which can target tumor vessels alternatively or even other components of the TME. This review aims to augment the current state of knowledge on VCO in CRCLM and other tumor entities and its impact on anti-angiogenic anti-cancer therapy.

Extracellular matrix scaffolds derived from different musculoskeletal tissues drive distinct macrophage phenotypes and direct tissue-specific cellular differentiation

The host immune response, specifically macrophage function, is a critical determinant of biomaterial success or failure post-implantation. Extracellular matrix (ECM) derived scaffolds have been shown to promote a pro-regenerative macrophage phenotype and a more constructive remodelling outcome. Here we demonstrate that macrophages adopt distinct phenotypes when exposed to articular cartilage (AC), ligament (LIG) and growth plate (GP) derived ECM scaffolds. Macrophages were generally unresponsive to LIG-derived ECM, adopted an M2-like phenotype when exposed to AC-derived ECM, and a hybrid M1-M2 phenotype when exposed to GP-ECM. Furthermore, macrophages expressed higher levels of pro-chondrogenic factors, such as FGF2, when exposed to AC-ECM, and higher levels of angiogenic and pro-osteogenic factors, such as VEGF, IL-6 and TNF, when exposed to GP-ECM. In addition, we observed that they can differentially direct the differentiation of skeletal stem cells, whereby AC-ECM promotes the chondrogenic differentiation and GP-ECM the osteogenic differentiation of multipotent stem/stromal cells (MSCs). In vivo characterisation of immune cell subsets following scaffold implantation into a large bone defect demonstrated that AC-ECM drives an M2 macrophage phenotype, while GP-ECM containing scaffolds promoted a hybrid M1-M2 phenotype and enhanced vascularisation and vessel maturation. This distinct response to the implantation of GP-ECM containing scaffolds was associated with increased CD45+ leukocyte and CD3+ T cell infiltration, accompanied by elevated concentrations of IFN-γ and IL-17. Taken together this work demonstrates that the source tissue of ECM scaffolds plays a key role in regulating the phenotype of both macrophages and skeletal stem cells. Furthermore, these ECMs can direct the cellular differentiation and production of growth factors essential for the regeneration of their source tissue. This work highlights the need for a more thorough characterisation of innate immune cell subsets post-biomaterial implantation.

Pulmonary Vascular Complications in Hereditary Hemorrhagic Telangiectasia and the Underlying Pathophysiology.

In this review, we discuss the role of transforming growth factor-beta (TGF-β) in the development of pulmonary vascular disease (PVD), both pulmonary arteriovenous malformations (AVM) and pulmonary hypertension (PH), in hereditary hemorrhagic telangiectasia (HHT). HHT or Rendu-Osler-Weber disease is an autosomal dominant genetic disorder with an estimated prevalence of 1 in 5000 persons and characterized by epistaxis, telangiectasia and AVMs in more than 80% of cases, HHT is caused by a mutation in the ENG gene on chromosome 9 encoding for the protein endoglin or activin receptor-like kinase 1 (ACVRL1) gene on chromosome 12 encoding for the protein ALK-1, resulting in HHT type 1 or HHT type 2, respectively. A third disease-causing mutation has been found in the SMAD-4 gene, causing a combination of HHT and juvenile polyposis coli. All three genes play a role in the TGF-β signaling pathway that is essential in angiogenesis where it plays a pivotal role in neoangiogenesis, vessel maturation and stabilization. PH is characterized by elevated mean pulmonary arterial pressure caused by a variety of different underlying pathologies. HHT carries an additional increased risk of PH because of high cardiac output as a result of anemia and shunting through hepatic AVMs, or development of pulmonary arterial hypertension due to interference of the TGF-β pathway. HHT in combination with PH is associated with a worse prognosis due to right-sided cardiac failure. The treatment of PVD in HHT includes medical or interventional therapy.

Phenotypic Modulation of Macrophages and Vascular Smooth Muscle Cells in Atherosclerosis-Nitro-Redox Interconnections.

Monocytes/macrophages and vascular smooth muscle cells (vSMCs) are the main cell types implicated in atherosclerosis development, and unlike other mature cell types, both retain a remarkable plasticity. In mature vessels, differentiated vSMCs control the vascular tone and the blood pressure. In response to vascular injury and modifications of the local environment (inflammation, oxidative stress), vSMCs switch from a contractile to a secretory phenotype and also display macrophagic markers expression and a macrophagic behaviour. Endothelial dysfunction promotes adhesion to the endothelium of monocytes, which infiltrate the sub-endothelium and differentiate into macrophages. The latter become polarised into M1 (pro-inflammatory), M2 (anti-inflammatory) or Mox macrophages (oxidative stress phenotype). Both monocyte-derived macrophages and macrophage-like vSMCs are able to internalise and accumulate oxLDL, leading to formation of “foam cells” within atherosclerotic plaques. Variations in the levels of nitric oxide (NO) can affect several of the molecular pathways implicated in the described phenomena. Elucidation of the underlying mechanisms could help to identify novel specific therapeutic targets, but to date much remains to be explored. The present article is an overview of the different factors and signalling pathways implicated in plaque formation and of the effects of NO on the molecular steps of the phenotypic switch of macrophages and vSMCs.

Abundance of Microvascular Endothelial Cells Is Associated with Response to Chemotherapy and Prognosis in Colorectal Cancer

Simple SummaryThe generation of pathologic, immature, and dysfunctional vessels by angiogenesis is a well-known mechanism of metastasis and has been a therapeutic target for colorectal cancer (CRC). In this study, we investigated the clinical relevance of microvascular endothelial (mvE) cells in CRC by analyzing tumor gene expression profiles of large patient cohorts. We found that the abundance of mvE cells does not mirror angiogenesis, but rather is associated with the number of mature blood vessels in the tumor microenvironment and predicts the response to chemotherapy as well as patient survival in CRC. This is the first study suggesting the clinical relevance of mvE cells in CRC.The generation of pathologic, immature, and dysfunctional vessels by angiogenesis is a mechanism of metastasis that has been a therapeutic target for colorectal cancer (CRC). In this study, we investigated the clinical relevance of intra-tumoral microvascular endothelial (mvE) cells in CRC using the xCell algorithm on transcriptome. A total of 1244 CRC patients in discovery and validation cohorts were analyzed. We found that an abundance of mvE cells did not mirror angiogenesis but reflected mature blood vessels because it was significantly associated with a high expression of vascular stability-related genes, including sphingosine-1-phosphate receptor genes and pericytes. Epithelial–mesenchymal transition and myogenesis gene sets were enriched in mvE cell abundant CRC, while mvE cell-less CRC enriched cell proliferation, oxidative phosphorylation, and protein secretion gene sets. mvE cell abundant CRC was associated with infiltration of M2 macrophages, dendritic cells, and less gamma-delta T cells (all p < 0.001), but not with the interferon-γ response. mvE cell abundant CRC was significantly associated with worse patient survival in CRC. Interestingly, mvE cell abundant CRC was significantly associated with a high response rate to chemotherapy (p = 0.012) and worse patient survival for those that did not receive chemotherapy. However, there was no survival difference in patients who underwent chemotherapy. In conclusion, we estimated the abundance of mvE cells using the xCell algorithm on tumor transcriptome finding its association with the number of mature blood vessels in a tumor microenvironment and its ability to predict response to chemotherapy, thereby patient survival in CRC.

Artificial Intelligence for ROP Screening and to Assess Quality of Care: Progress and Challenges.

The goal of retinopathy of prematurity (ROP) screening is to detect the constellation of clinical signs that suggest a high risk of progression to retinal detachment so that urgent treatment can be given. At each screening episode, there are 3 considerations: whether urgent treatment is needed, whether follow-up screening is needed, and whether there is no risk of sight-threatening ROP so that the screening can stop. The decisions are based on a detailed examination of the retina focused on the severity (ie, stage), location (ie, zone), and degree of dilation and tortuosity of retinal blood vessels (ie, plus disease). If ROP is not present, the peripheral retina needs to be assessed to determine the degree of maturity of the retinal vessels; if they are mature or ROP detected earlier is definitely regressing, the screening can stop.Telemedicine with artificial intelligence (AI) image analysis could transform ROP screening, especially in settings with an insufficient supply of ophthalmologists, which can happen either because of absolute workforce shortages or when ophthalmologists choose not to offer ROP screening because of the time burden or fear of litigation. Telemedicine with AI image analysis is particularly appealing for low-resource settings in which greater exposure to risk factors, such as hyperoxia, can lead to a high frequency of treatment-requiring ROP in infants born at a gestational age (GA) of <32 weeks, and severe disease can also develop in more mature infants.1 In these settings, wider screening criteria are required, which increases the screening workload.2In this issue of Pediatrics, Campbell et al3 describe a multicenter screening program in India, the Retinopathy of Prematurity Eradication Save Our Sight program, which was done in collaboration with a United States–based international group, Imaging and Informatics in Retinopathy of Prematurity (iROP). This report describes not only the diagnostic-test accuracy of the deep learning (DL) system developed by this group (iROP DL) but, also, whether the findings can be used as an indicator to assess the quality of neonatal care.iROP DL, which quantifies plus disease, performed well, with 100% sensitivity and a high specificity compared to image grading by 3 ROP experts as the gold standard. Because missing ROP that requires treatment can have serious vision consequences, a high sensitivity is essential. The low positive predictive value is not surprising because of the low prevalence of treatment-requiring ROP.The study findings related to quality of care are particularly interesting. There were highly significant associations between the mean iROP DL score and lower birth weight and whether the unit had adequate equipment to mix and monitor oxygen. A striking finding was that only 3 of the 14 study units had enough of this equipment, which is a frequent and regrettable occurrence in a large number of units in many low- and middle-income countries.There are some important limitations of this study. iROP cannot be used to assess stage or zone, only plus disease. Whether DL and AI image analysis can achieve equally high diagnostic-test accuracy for these other prognostic signs, compared to clinical examination by indirect ophthalmoscopy or image analysis by an ROP expert, remains to be seen. In India and other low-resource settings, data on GA can be unreliable because of low attendance at antenatal care, high rates of intrauterine-growth restriction, and lack of routine prenatal ultrasound. Because GA is an important risk factor for ROP, in this study, more accurate data may have attenuated the association between iROP DL scores and quality of care. The study findings might not be generalizable to other regions. For example, the degree of retinal pigmentation, which can be greater for children born in Africa, could impact the interpretation of images.Several other groups are also working on AI for ROP screening,4–7 and once systems can provide reliable information on all of the signs needed to make a management decision at each screening episode, together with the availability of lower-cost imaging devices, the door is open for a dramatic change in how ROP screening is delivered and by whom.8In 2019, the World Health Organization and United Nations Children’s Fund recommended ROP screening and treatment as standard of care in their “Survive and Thrive” policy.9 The authors correctly indicate that integrated systems will be essential before the benefits of telemedicine and AI image analysis can be fully realized. Studies of the feasibility, acceptability, and cost-effectiveness of this approach would also be required for decision-makers and policy change within countries, with allocation of resources for implementation.

Relating prostate‐specific antigen leakage with vascular tumor growth in a mathematical model of prostate cancer response to androgen deprivation

AbstractThe use of prostate‐specific antigen (PSA) as a prognostic indicator for prostate cancer (PCa) patients is controversial, especially since it has been shown to correlate poorly with tumor burden. The poor quality of PSA as a biomarker could be explained by current guidelines not accounting for the mechanism by which it enters circulation. Given that mature blood vessels are relatively impermeable to it, we hypothesize that immature and leaky blood vessels, formed under angiogenic cues in a hypoxic tumor, facilitate PSA extravasation into circulation. To explore our hypothesis, we develop a nonlinear dynamical systems model describing the vascular growth of PCa, that explicitly links PSA leakage into circulation with changes in intra‐tumoral oxygen tension and vessel permeability. The model is calibrated versus serum PSA and tumor burden time‐courses from a mouse xenograft model of castration resistant PCa response to androgen deprivation. The model recapitulates the experimentally observed and – counterintuitive – phenomenon of increasing tumor burden despite decreasing serum PSA levels. The validated model is then extended to the human scale by incorporating patient‐specific parameters and fitting individual PSA time‐courses from patients with biochemically failing PCa. Our results highlight the limitations of using time to PSA failure as a clinical indicator of androgen deprivation efficacy. We propose an alternative indicator, namely a treatment efficacy index, for patients with castration resistant disease, to identify who would benefit most from enhanced androgen deprivation. A critical challenge in PCa therapeutics is quantifying the relationship between serum PSA and tumor burden. Our results underscore the potential of mathematical modeling in understanding the limitations of serum PSA as a prognostic indicator. Finally, we provide a means of augmenting PSA time‐courses in the diagnostic process, with changes in intra‐tumoral vascularity and vascular architecture.

The effects of vascularization on tumor development: A systematic review and meta-analysis of pre-clinical studies

This review aimed to systematize and quantify the existing evidence about the effect of tumor vascularization on its growth, in preclinical studies. A computerized research on databases PubMed, Scopus and EBSCO was performed to identify studies that evaluate both the vascularization parameters and the development of the tumors in animal models and the mean differences were calculated through a random effects model. Thirteen studies met the inclusion criteria and were included in the systematic review, of which, 6 studies were included in the meta-analysis. Besides tumor vascular density that all studies evaluated, 3 studies analysed the tumor perfusion, 2 studies the tumor hypoxia and 3 studies assessed the grade of vessel maturation. Most of the studies (11) related decreased tumor vascularization and a concomitant inhibition of tumor growth or metastasis development. Quantitatively, the decrease in tumor vascularization contributed to a significant decrease in the tumor growing rate of 5.23 (-9.20, -1.26). A reduced level of tumor vascularization seems to be able to inhibit tumor growth and progression.

Atorvastatin pleiotropically decreases intraplaque angiogenesis and intraplaque haemorrhage by inhibiting ANGPT2 release and VE-Cadherin internalization

ObjectiveStatins pleiotropically provide additional benefits in reducing atherosclerosis, but their effects on intraplaque angiogenesis (IPA) and hemorrhage (IPH) remain unclear. Therefore, we discriminated statin’s lipid-lowering dependent and independent effects on IPA and IPH.Approach and resultsApoE3*Leiden mice are statin-responsive due to ApoE and LDLR presence, but also allow to titrate plasma cholesterol levels by diet. Therefore, ApoE3*Leiden mice were fed a high-cholesterol-inducing-diet (HCD) with or without atorvastatin (A) or a moderate-cholesterol-inducing-diet (MCD). Mice underwent vein graft surgery to induce lesions with IPA and IPH. Cholesterol levels were significantly reduced in MCD (56%) and HCD + A (39%) compared to HCD with no significant differences between MCD and HCD + A. Both MCD and HCD + A have a similar reduction in vessel remodeling and inflammation comparing to HCD. IPA was significantly decreased by 30% in HCD + A compared to HCD or MCD. Atorvastatin treatment reduced the presence of immature vessels by 34% vs. HCD and by 25% vs. MCD, resulting in a significant reduction of IPH. Atorvastatin’s anti-angiogenic capacity was further illustrated by a dose-dependent reduction of ECs proliferation and migration. Cultured mouse aortic-segments lost sprouting capacity upon atorvastatin treatment and became 30% richer in VE-Cadherin expression and pericyte coverage. Moreover, Atorvastatin inhibited ANGPT2 release and decreased VE-Cadherin(Y685)-phosphorylation in ECs.ConclusionsAtorvastatin has beneficial effects on vessel remodeling due to its lipid-lowering capacity. Atorvastatin has strong pleiotropic effects on IPA by decreasing the number of neovessels and on IPH by increasing vessel maturation. Atorvastatin improves vessel maturation by inhibiting ANGPT2 release and phospho(Y658)-mediated VE-Cadherin internalization.

A Comparison of the Accuracy of Various Methods of Postnatal Gestational Age Estimation; Including Ballard Score, Foot Length, Vascularity of the Anterior Lens, Last Menstrual Period and Also a Clinician's Non-Structured Assessment.

Gestational age is a strong determinant of neonatal mortality and morbidity. Early obstetric ultrasound is the clinical reference standard, but is not widely available in many developing countries. A prospectively designed diagnostic accuracy study in a tertiary referral hospital in a developing country. Early ultrasound (<20 weeks) was the clinical reference standard. Methods evaluated included anthropometric measurements (including foot length), vascularity of the anterior lens, the New Ballard Score and last menstrual period. Clinicians' non-structured global impression 'End of Bed' Assessment was also evaluated. 106 babies were included in the study. Median age at birth was 34 weeks (interquartile range 29-36). Ballard Score and 'End of Bed' Assessment had a mean bias of -0.14 and 0.06 weeks respectively but wide 95% limits of agreement. The physical component of the Ballard score, the total Ballard score and Foot length's ability to discriminate between term and preterm infants gave an area under the receiver operating characteristics curve of 0.97, 0.96 and 0.95, respectively. Although 'End of Bed' Assessment and Ballard score had small mean biases, the wide confidence intervals render the methods irrelevant in clinical practice. Foot length was particularly poor in Small for Gestational Age infants. None of the methods studied were superior to a non-structured clinician's informal 'End of Bed' Assessment. None of the methods studied met the a priori definition of clinical usefulness. Improving access to early ultrasound remains a priority. Instead of focusing on chronological accuracy, future research should compare the ability of early ultrasound and Ballard score to predict morbidity and mortality. Lay summary. Gestational age describes the time interval between conception and the delivery of the baby. Babies born before 37 weeks of gestation (preterm) or after 42 weeks of gestation (post-dates) have an increased risk of death and specific illnesses. The best way to estimate the gestational age is to perform an ultrasound scan on the mother before 20 weeks. However, this is not widely available in many developing countries. Methods to estimate gestational age after birth include calculating the time from the last period, various measurements of the child (such as weight, foot length or head circumference) physical and neurological markers of maturity and examination of the blood vessels on the lens in the eye. In this study, we assessed how accurate these methods were when compared with the best available method; early ultrasound. We also analyzed the clinicians own personal feeling of what the most likely gestation was, based on an informal 'end of bed' assessment. If a method was to be deemed clinically useful it was agreed that it would have to confidently identify the gestation to within 1 week of the true gestation. None of the methods studied could confidently predict the gestational age of individual babies within 1 week. Ballard scoring and the clinician's informal 'End of Bed' Assessment were the most accurate and also had the smallest inter-operator variability when the results of two separate researchers were compared. Foot length performed particularly badly with babies who were small for their gestational age. None of the methods studied confidently predicted gestational age within a week, so have little use in clinical practice. Access to early ultrasound should be improved. Further research into the relationship between maturity markers such as the Ballard score and the rates of death and specific premature related illnesses is warranted.

Tumor Hypoxia Regulates Immune Escape/Invasion: Influence on Angiogenesis and Potential Impact of Hypoxic Biomarkers on Cancer Therapies.

The environmental and metabolic pressures in the tumor microenvironment (TME) play a key role in molding tumor development by impacting the stromal and immune cell fractions, TME composition and activation. Hypoxia triggers a cascade of events that promote tumor growth, enhance resistance to the anti-tumor immune response and instigate tumor angiogenesis. During growth, the developing angiogenesis is pathological and gives rise to a haphazardly shaped and leaky tumor vasculature with abnormal properties. Accordingly, aberrantly vascularized TME induces immunosuppression and maintains a continuous hypoxic state. Normalizing the tumor vasculature to restore its vascular integrity, should hence enhance tumor perfusion, relieving hypoxia, and reshaping anti-tumor immunity. Emerging vascular normalization strategies have a great potential in achieving a stable normalization, resulting in mature and functional blood vessels that alleviate tumor hypoxia. Biomarkers enabling the detection and monitoring of tumor hypoxia could be highly advantageous in aiding the translation of novel normalization strategies to clinical application, alone, or in combination with other treatment modalities, such as immunotherapy.

Mesenchymal glioblastoma-induced mature de-novo vessel formation of vascular endothelial cells in a microfluidic device

High vascularization is a biological characteristic of glioblastoma (GBM); however, an in-vitro experimental model to verify the mechanism and physiological role of vasculogenesis in GBM is not well-established. Recently, we established a self-organizing vasculogenic model using human umbilical vein endothelial cells (HUVECs) co-cultivated with human lung fibroblasts (hLFs). Here, we exploited this system to establish a realistic model of vasculogenesis in GBM. We developed two polydimethylsiloxane (PDMS) devices, a doughnut-hole dish and a 5-lane microfluidic device to observe the contact-independent effects of glioblastoma cells on HUVECs. We tested five patient-derived and five widely used GBM cell lines. Confocal fluorescence microscopy was used to observe the morphological changes in Red Fluorescent Protein (RFP)-HUVECs and fluorescein isothiocyanate (FITC)-dextran perfusion. The genetic and expression properties of GBM cell lines were analyzed. The doughnut-hole dish assay revealed KNS1451 as the only cells to induce HUVEC transformation to vessel-like structures, similar to hLFs. The 5-lane device assay demonstrated that KNS1451 promoted the formation of a vascular network that was fully perfused, revealing the functioning luminal construction. Microarray analysis revealed that KNS1451 is a mesenchymal subtype of GBM. Using a patient-derived mesenchymal GBM cell line, mature de-novo vessel formation could be induced in HUVECs by contact-independent co-culture with GBM in a microfluidic device. These results support the development of a novel in vitro research model and provide novel insights in the neovasculogenic mechanism of GBM and may potentially facilitate the future detection of unknown molecular targets.