Neovascular Response Research Articles

Lymphatic-macrophage crosstalk during neonatal mouse heart regeneration and transition to fibrotic repair

Abstract Background In adult mice, myocardial infarction (MI) activates the cardiac lymphatics, which undergo sprouting angiogenesis (lymphangiogenesis) and function to drain interstitial fluid and traffic macrophages to mediastinal lymph nodes (MLNs). This prevents oedema and reduces inflammatory immune cell content to improve cardiac function. Purpose Given the importance of the adult cardiac lymphatics in macrophage clearance after injury, we investigated their role across the neonatal "regenerative window". At post-natal day 1 (P1) neonatal mice fully regenerate their heart following MI, in a macrophage-dependent manner, whereas equivalent injury at day 7 (P7) leads to scarring driven by pro-fibrotic macrophages. We hypothesised that lymphatics respond and function differently during this "window," to clear macrophage-specific subtypes depending upon their requirement for regeneration versus fibrotic repair. Methods & Results Extensive lymphatic growth and sprouting was evident in intact neonatal hearts until P16, with strain-dependent developmental differences. The response to injury revealed limited lymphangiogenesis and minimal clearance of macrophages from P1 compared to P7 infarcted hearts, as determined by adoptive transfer experiments and monitoring of cleared macrophages to MLNs. This is coincident with maturation of lymphatic endothelial cell junctions across the neonatal period via transition from "zipper" (impermeable) to "button" (permeable) -type junctions. To gain molecular insight into the mechanisms underpinning lymphatic endothelium macrophage interactions at P1 versus P7, we generated unbiased single cell RNA sequencing datasets from neonatal heart samples after MI and observed altered signalling between lymphatic endothelial cells and macrophages across the neonatal period, including altered expression of the lymphangiocrine factor Reelin (RELN). Finally, in mice lacking the lymphatic endothelial receptor-1 (LYVE1), that exhibit impaired transmigration of interstitial macrophages to lymphatic vessels, magnetic resonance imaging (MRI) revealed a surprising impaired functional outcome in P1 mice 28 days post-MI. Given our observations that pro-regenerative macrophages at P1 are not trafficked, this suggested a distinct role for LYVE1 in tissue-resident (TR) macrophages, consistent with its expression in developing and post-natal hearts. Macrophage-specific deletion of Lyve1 during neonatal heart injury revealed impaired heart regeneration, characterised by reduced neovascular response and function, suggesting a hitherto unappreciated role for LYVE1 in regulating the pro-regenerative function of TR macrophages. Conclusions Collectively, we reveal that cardiac lymphatics are developmentally compromised for immune cell clearance in early neonates, which enables retention of pro-regenerative TR macrophages, and that LYVE1 plays an essential role in TR macrophages enabling heart regeneration via the induction of coronary angiogenesis.

The Pan-Tumor Vasculature under the Transcriptomic Magnifying Glass.

In the first pan-cancer analysis of the tumor vasculature, Pan and colleagues profile nearly 200,000 endothelial cells (EC) and mural cells, identifying novel subclusters and cell states using consensus trajectory inference. They identify differentiation trajectories in vascular and lymphatic ECs and subtype the pericyte (PC) population. During sprouting angiogenesis, venous cells dedifferentiate and transition to capillary and, finally, arterial ECs. Capillary ECs transition via "three angiogenic stages" (SI-SIII), during which APLN+ TipS1 cells were identified as potential modulators of tumor-induced neovascularization and antiangiogenic therapy response. In lymphatic ECs, differentiation was inversely correlated between the lymphangiogenic (T1) and antigen-presenting (T2) trajectories, with T2 associated with a better prognosis. Although several PC clusters were identified, BASP1+ matrix-associated PCs were associated with APLN+ TipS1 cells and had a worse prognosis. These findings present transcriptional validation of previous experimental findings and serve as a resource to examine the tumor vascular microenvironment in detail.

PolySialic Acid Nanoparticles Actuate Complement-Factor-H-Mediated Inhibition of the Alternative Complement Pathway: A Safer Potential Therapy for Age-Related Macular Degeneration.

The alternative pathway of the complement system is implicated in the etiology of age-related macular degeneration (AMD). Complement depletion with pegcetacoplan and avacincaptad pegol are FDA-approved treatments for geographic atrophy in AMD that, while effective, have clinically observed risks of choroidal neovascular (CNV) conversion, optic neuritis, and retinal vasculitis, leaving room for other equally efficacious but safer therapeutics, including Poly Sialic acid (PSA) nanoparticle (PolySia-NP)-actuated complement factor H (CFH) alternative pathway inhibition. Our previous paper demonstrated that PolySia-NP inhibits pro-inflammatory polarization and cytokine release. Here, we extend these findings by investigating the therapeutic potential of PolySia-NP to attenuate the alternative complement pathway. First, we show that PolySia-NP binds CFH and enhances affinity to C3b. Next, we demonstrate that PolySia-NP treatment of human serum suppresses alternative pathway hemolytic activity and C3b deposition. Further, we show that treating human macrophages with PolySia-NP is non-toxic and reduces markers of complement activity. Finally, we describe PolySia-NP-treatment-induced decreases in neovascularization and inflammatory response in a laser-induced CNV mouse model of neovascular AMD. In conclusion, PolySia-NP suppresses alternative pathway complement activity in human serum, human macrophage, and mouse CNV without increasing neovascularization.

Single-nucleus expression characterization of non-enhancing region of recurrent high-grade glioma.

Non-enhancing (NE) infiltrating tumor cells beyond the contrast-enhancing (CE) bulk of tumor are potential propagators of recurrence after gross total resection of high-grade glioma. We leveraged single-nucleus RNA sequencing on 15 specimens from recurrent high-grade gliomas (n = 5) to compare prospectively identified biopsy specimens acquired from CE and NE regions. Additionally, 24 CE and 22 NE biopsies had immunohistochemical staining to validate RNA findings. Tumor cells in NE regions are enriched in neural progenitor cell-like cellular states, while CE regions are enriched in mesenchymal-like states. NE glioma cells have similar proportions of proliferative and putative glioma stem cells relative to CE regions, without significant differences in % Ki-67 staining. Tumor cells in NE regions exhibit upregulation of genes previously associated with lower grade gliomas. Our findings in recurrent GBM paralleled some of the findings in a re-analysis of a dataset from primary GBM. Cell-, gene-, and pathway-level analyses of the tumor microenvironment in the NE region reveal relative downregulation of tumor-mediated neovascularization and cell-mediated immune response, but increased glioma-to-nonpathological cell interactions. This comprehensive analysis illustrates differing tumor and nontumor landscapes of CE and NE regions in high-grade gliomas, highlighting the NE region as an area harboring likely initiators of recurrence in a pro-tumor microenvironment and identifying possible targets for future design of NE-specific adjuvant therapy. These findings also support the aggressive approach to resection of tumor-bearing NE regions.

Exosomes: potential diagnostic markers and drug carriers for adenomyosis.

Adenomyosis is a common benign gynecological disorder and an important factor leading to infertility in fertile women. Adenomyosis can cause deep lesions and is persistent and refractory in nature due to its tumor-like biological characteristics, such as the ability to implant, adhere, and invade. The pathogenesis of adenomyosis is currently unclear. Therefore, new therapeutic approaches are urgently required. Exosomes are nanoscale vesicles secreted by cells that carry proteins, genetic materials and other biologically active components. Exosomes play an important role in maintaining tissue homeostasis and regulating immune responses and metabolism. A growing body of work has shown that exosomes and their contents are key to the development and progression of adenomyosis. This review discusses the current research progress, future prospects and challenges in this emerging therapeutic tool by providing an overview of the changes in the adenomyosis uterine microenvironment and the biogenesis and functions of exosomes, with particular emphasis on the role of exosomes and their contents in the regulation of cell migration, proliferation, fibrosis formation, neovascularization, and inflammatory responses in adenomyosis.

Stimulation of soluble guanylate cyclase activity with riociguat promotes angiogenesis and improves neovascularization after limb ischemia

Background and aimsThe NO-cGMP pathway is essential for angiogenesis, vasculogenesis and post-natal neovascularization. The key enzyme responsible for the synthesis of cGMP following binding of NO is soluble guanylate cyclase (sGC). Riociguat is the first member of a novel class of compounds known as sGC stimulators. We tested the hypothesis that stimulation of sGC with riociguat might improve neovascularization in response to ischemia. MethodsIn vitro, the angiogenic effect of riociguat was tested in human umbilical vein endothelial cells (HUVECs). In vivo, neovascularization was investigated in a mouse model of limb ischemia. C57Bl/6 mice were treated by gavage with 3 mg/kg/day of riociguat for a total of 28 days. After two weeks of treatment, hindlimb ischemia was surgically induced by femoral artery removal. ResultsIn a matrigel assay in vitro, riociguat dose-dependently stimulates tubule formation in HUVECs. Cell migration (scratch assay) is also increased in HUVECs treated with riociguat. At the molecular level, riociguat treatment leads to rapid activation of the p44/p42 MAP kinase pathway in HUVECs. Inhibition of protein kinase G (PKG) activity supresses both p44/p42 MAP kinase activation and angiogenesis in HUVECs treated with riociguat. In vivo, treatment with riociguat improves blood flow recovery after ischemia (Laser Doppler imaging), and increases capillary density in ischemic muscles (CD31 immunostaining). Clinically, this is associated with a significant decrease of ambulatory impairment and ischemic damages. Interestingly, mice treated with riociguat also show a 94% increase in the number of bone marrow-derived pro-angiogenic cells (PACs) compared to control mice. Moreover, riociguat treatment is associated with a significant improvement of PAC functions including migratory capacity, adhesion to an endothelial monolayer, and integration into endothelial tubular networks. ConclusionsThe sGC stimulator riociguat promotes angiogenesis and improves neovascularization after ischemia. The mechanism involves PKG-dependent activation of p44/p42 MAP kinase pathway, together with an improvement of PAC number and functions. sGC stimulation could constitute a novel therapeutic strategy to reduce tissue ischemia in patients with severe atherosclerotic diseases.

Synthesis of oxidized sodium alginate and its electrospun bio-hybrids with zinc oxide nanoparticles to promote wound healing

Electrospun fibers provide a promising platform for wound healing; however, they lack requisite characteristics for wound repair, including antibacterial and anti-inflammatory properties and angiogenic ability. Sodium alginate (SA) is being used for different types of applications. However, the poor spinnability of SA restricts its applications. The objectives of this study were three-fold: a) to synthesize oxidized sodium alginate (OSA) to improve its spinnability, b) to fabricate composite fibrous membranes by blending OSA along with zinc oxide nanoparticles (ZnO-NPs), and c) to decipher antibacterial and anti-inflammatory properties as well as biocompatibility of membranes in vitro and in vivo. OSA displaying different oxidation degrees (Dox (%)) was synthesized by varying the molar ratio of sodium periodate to SA. OSA (Dox, ∼48 %) afforded smooth and uniform fibers; 0.5 wt% of adipic dihydrazide (ADH) evolved into structurally stable and water-insoluble membranes. Composite fibrous membranes containing 2 wt% of ZnO-NPs displayed good biocompatibility and bactericidal effect against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) in vitro. In addition, composite membranes showed remarkable epithelialization, neovascularization, and anti-inflammatory response than that of the membranes devoid of ZnO-NPs. Conclusively, these composite fibrous membranes may have broad implications for wound healing applications.

Evaluation of the role of optical coherence tomography angiography in monitoring treatment response in patients with neovascular age-;1;related macular degeneration

Aim The aim was to assess the role of optical coherence tomography angiography (OCTA) in the diagnosis, assessment of activity, and monitoring the treatment response of choroidal neovascularization secondary to age-related macular degeneration (AMD). Methods Prospective, interventional case series of eyes that were diagnosed with active neovascular AMD. Spectral-domain optical coherence tomography (SD-OCT) and OCTA were done at baseline after intravitreal antivascular endothelial growth factor (anti-VEGF) treatment to determine OCTA sensitivity in the detection of activity in relation to SD-OCT findings and its specificity following intervention. Results Twenty-five eyes were included, of which 20 eyes were imaged successfully by OCTA. The diagnostic sensitivity of OCTA in those eyes was found to be 75%, and its sensitivity in the assessment of neovascular activity was 80 and 60%, before and after anti-VEGF therapy, respectively, whereas its specificity after anti-VEGFs therapy was 100%. Conclusion Although SD-OCT continues to be the gold-standard for noninvasively diagnosing and tracking neovascular AMD treatment response, OCTA may offer a noninvasive option that can support treatment selection throughout follow-up and guide efficient therapeutic approaches. Author contributions: All authors contributed to the construction of idea and question of the research with complete assessment and managements of all the study group. Hebatalla S. Makled: Assessment of the patients with follow-up, doing OCTA for the patients with interpretation, data collection and analysis, and manuscript writing and revision. Ahmad Almabrook Sahban: recruitment of patients with consent acquisition, doing OCTA for the patients, treatment with follow-up of the patients, collection and analysis of data, and manuscript writing. Ayman M Khattab: Patients assessment, decision making and follow-up of patients treatment, OCTA interpretation, collection and analysis of data, and manuscript writing and revision. Ashraf Ahmed Nossair: Patients assessment with follow-up, OCTA interpretation, data collection and analysis, and manuscript writing and revision.

Combined Anti-Angiogenic and Anti-Inflammatory Nanoformulation for Effective Treatment of Ocular Vascular Diseases.

Ocular vascular diseases are the major causes of visual impairment, which are characterized by retinal vascular dysfunction and robust inflammatory responses. Traditional anti-angiogenic or anti-inflammatory drugs still have limitations due to the short-acting effects. To improve the anti-angiogenic or anti-inflammatory efficiency, a dual-drug nanocomposite formulation was proposed for combined anti-angiogenic and anti-inflammatory treatment of ocular vascular diseases. CBC-MCC@hMSN(SM) complex nanoformulation was prepared by integrating conbercept (CBC, an anti-angiogenic drug) and MCC950 (MCC, an inhibitor of inflammation) into the surface-modified hollow mesoporous silica nanoparticles (hMSN(SM)). CBC-MCC@hMSN(SM) complex nanoformulation was then characterized by Fourier transform infrared spectroscopy, transmission electron microscopy, zeta potentials, and nitrogen adsorption-desorption measurement. CBC and MCC release profile, cytotoxicity, tissue toxicity, anti-angiogenic effects, and anti-inflammatory effects of CBC-MCC@hMSN(SM) were estimated using the in vitro and in vivo experiments. CBC-MCC@hMSN(SM) complex had no obvious cytotoxicity and tissue toxicity and did not cause a detectable ocular inflammatory responses. CBC-MCC@hMSN(SM) complex was more effective than free CBC or MCC in suppressing endothelial angiogenic effects and inflammatory responses in vitro. A single intraocular injection of CBC-MCC@hMSN(SM) complex potently suppressed diabetes-induced retinal vascular dysfunction, choroidal neovascularization, and inflammatory responses for up to 6 months. Combined CBC and MCC nanoformulation provides a promising strategy for sustained suppression of pathological angiogenesis and inflammatory responses to improve the treatment outcomes of ocular vascular diseases.

Abstract 14936: Profiling of Microrna Expression in Extracellular Vesicles Released From Ischemic Skeletal Muscles in Hypercholesterolemic Conditions

Introduction: Hypercholesterolemia (HC) is an important cardiovascular risk factor associated with impaired neovascularization in response to ischemia. We found that specific microRNAs (miRs) whose levels are reduced in ischemic skeletal muscles of mice exposed to HC can rescue endothelial cell function and neovascularization. miRs can be transferred between cells via extracellular vesicles (EVs). EVs are involved in several biological functions including the response to ischemia and angiogenesis. Hypothesis: Here we tested the hypothesis that HC causes alterations in the miR content of EVs related to ischemia/neovascularization. Methods: We used a mouse model of peripheral artery disease to induce severe ischemia in the hindlimbs of hypercholesterolemic ApoE -/- and control C57Bl/6J mice. Microvesicles (MVs) and exosomes (exo) were isolated from ischemic skeletal muscles by differential centrifugation. Total RNA was extracted from these 2 types of EVs, and the miR content was analyzed using next generation sequencing. Results: Bioinformatic analysis of the 100 most expressed miRs in EVs showed an enrichment of 38 miRs in MVs compared to ischemic skeletal muscles, and an enrichment of 44 miRs in exo compared to ischemic skeletal muscles. Among enriched miRs, 27 are common to both MVs and exo. We identified specific miRs enriched in EVs that are known to modulate angiogenesis, including several miRs that were altered by HC. For example, among enriched miRs downregulated by HC in EVs, the let-7 family and miR-29a are known to be pro-angiogenic via inhibition of members of the TIMP family. On the other hand, among enriched miRs upregulated by HC in EVs, miR-199a and miR-16-5p are known to be anti-angiogenic via inhibition of VEGF. Conclusions: This study describes for the first time the effect of HC on the modulation of miR profile in EVs following skeletal muscle ischemia. We found that several angiogenesis-modulating miRs are enriched in EVs and altered by HC. Our findings constitute a solid framework for the identification of miRs that could be targeted to modify EV content in atherosclerotic conditions. Eventually these engineered EVs could serve as vectors to promote neovascularization and reduce ischemic damages in severe vascular diseases.

Melatonin Improves Ischemia-Induced Circulation Recovery Impairment in Mice with Streptozotocin-Induced Diabetes by Improving the Endothelial Progenitor Cells Functioning.

Patients with diabetes mellitus tend to develop ischemia-related complications and have compromised endothelial progenitor cell (EPC) function. Melatonin protects against ischemic injury, possibly via EPC modulation. We investigated whether melatonin pretreatment could restore EPC function impairment and improve circulation recovery in a diabetic critical limb ischemia mouse model. Under 25 mM high-glucose medium in vitro, EPC proliferation, nitric oxide production, tube formation, and endothelial nitric oxide synthase (eNOS) phosphorylation were significantly suppressed. Hyperglycemia promoted EPC senescence and apoptosis as well as increased reactive oxygen species (ROS) production. Melatonin treatment reversed the harmful effects of hyperglycemia on EPC through adenosine monophosphate–activated protein kinase-related mechanisms to increase eNOS phosphorylation and heme oxygenase-1 expression. In an in-vivo study, after a 4-week surgical induction of hindlimb ischemia, mice with streptozotocin (STZ)-induced diabetes showed significant reductions in new vessel formation, tissue reperfusion, and EPC mobilization in ischemic hindlimbs compared to non-diabetic mice. Mice with STZ-induced diabetes that received melatonin treatment (10 mg/kg/day, intraperitoneal) had significantly improved blood perfusion ratios of ischemic to non-ischemic limb, EPC mobilization, and densities of capillaries. In addition, a murine bone marrow transplantation model to support these findings demonstrated that melatonin stimulated bone marrow-originated EPCs to differentiate into vascular endothelial cells in femoral ligation-induced ischemic muscles. In summary, this study suggests that melatonin treatment augments EPC function along with neovascularization in response to ischemia in diabetic mice. We illustrated the protective effects of melatonin on EPC H2O2 production, senescence, and migration through melatonin receptors and modulating eNOS, AMPK, and HO-1 activities at the cellular level. Thus, melatonin might be used to treat the impairment of EPC mobilization and circulation recuperation in response to ischemic injury caused by chronic hyperglycemia. Additional studies are needed to elucidate the applicability of the results in humans.

Myeloid cells regulate retinal neovascularization via SOCS3

Abstract Neovascularization is the leading cause of vision loss in retinopathy of prematurity (ROP). Inflammation and myeloid cells are involved in retinal angiogenesis. However, the underlying mechanisms of how myeloid cells control neovascularization in ROP remain unknown. We and others previously reported that suppressor of cytokine signaling 3 (SOCS3) is induced in retinopathy. To investigate whether myeloid cells regulate retinal angiogenesis via SOCS3 in ROP, we used myeloid lineage specific SOCS3 loss-of-function (conditional knockout, cKO) mice and gain-of-function (conditional overexpression, cOE) mice and generated ROP mouse model - mice with oxygen induced retinopathy (OIR) by exposing mice to 75% oxygen from postnatal day (P)7 to P12 for 5 days, and then returned to normal air until P17. Retinas were collected for analysis including quantification of retinal neovascularization, gene expression, immune cell profiling and single cell sequencing. Our results showed that mice with myeloid SOCS3 deficiency had a substantial increase in pathological retinal neovascularization, whereas overexpression of SOCS3 in myeloid cells prevented pathological retinal neovascularization in response to OIR. Our single-cell sequencing data showed that population of myeloid lineage cells including microglia, macrophages and neutrophils was changed in mice with OIR and controlled by SOCS3. Therefore, myeloid cells are involved in retinal neovascularization via SOCS3 in ROP. Manipulating SOCS3 expression in myeloid cells may provide a new way to cure neovascularization in retinas. Supported by grants from NIH (R01EY030140, R01EY029238) and from BrightFocus Foundation

Melatonin exerts anti-angiogenic and anti-inflammatory effects in alkali-burned corneas.

BackgroundCorneal neovascularization (CNV) caused by alkali burn injury is tightly associated with an inflammatory reaction and can lead to vision loss. Melatonin is involved in anti-inflammation and anti-angiogenesis, but its role in CNV has not yet been investigated.MethodsWe induced CNV using sodium hydroxide (NaOH) and compared the reactions of vehicle control and melatonin-treated male C57BL/6 mice at 7 and 14 days following the corneal burn. The infiltration of inflammatory cells and the expression of proangiogenic factors, chemokines, and inflammation-related molecules were quantified via immunohistochemical (IHC) analysis and quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR), respectively. Murine peritoneal macrophages were used in vitro to further verify the effect of melatonin in inflammatory CNV.ResultsCompared with the vehicle control mice, the melatonin-treated mice showed significant inhibition of angiogenesis and reduction of corneal epithelial defects in alkali-burned corneas. Concomitantly, the infiltration of inflammatory cells and F4/80+ cells were dramatically reduced after melatonin treatment. The messenger RNA (mRNA) expression of proangiogenic factors [vascular endothelial growth factors (VEGF) and matrix metalloproteinase-9 (MMP-9)], monocyte chemotactic protein-1 (MCP-1), nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3), and pro-inflammatory cytokines [tumor necrosis factor-alpha (TNF-α) and interleukin-1β (IL-1β) and IL-6] were down-regulated in the melatonin-treated mice. Moreover, melatonin inhibited the expression of these factors in murine peritoneal macrophages.ConclusionsMelatonin inhibits the neovascular and inflammatory responses in corneal alkali burn injury, suggesting that it may be a potential therapy for CNV.

Fluid dynamics between injections in incomplete anti-VEGF responders within neovascular age-related macular degeneration: a prospective observational study

BackgroundThe purpose of the study was to investigate the short-term response profile after an intravitreal injection (IVI) of anti-vascular endothelial growth factor (VEGF) in patients with neovascular age-related macular degeneration (nAMD) and incomplete response to anti-VEGF.MethodsIn this monocentric prospective observational study, we recruited patients with incomplete response to anti-VEGF, defined as presence of subretinal fluid (SRF) and/or intraretinal fluid (IRF) on optical coherence tomography (OCT) for at least 6 months despite monthly anti-VEGF treatment. Each patient underwent complete ophthalmic exam and imaging study (including OCT, fluorescein angiography, indocyanine green angiography, OCT-angiography) the day of their scheduled monthly IVI. Intermediate visits were performed weekly thereafter (comprising ophthalmic exam and OCT), until week 4. Fluid metrics were quantified using an artificial intelligence-based algorithm at baseline and at each subsequent weekly visit. Main outcomes were residual fluid volumes of SRF and IRF for each time point, and its relative change after treatment. Particular interest was given to each patients’ nadir point, which was used for association analysis with imaging parameters.ResultsA total of 28 eyes of 26 patients were included into the study. The maximal response was reached at 1.93 weeks on average. The relative fluid resolution at nadir point was 66 ± 36.7%, with quartile limits at 49.1%, 83%, and 96.1%, respectively. Mean residual fluid volume was 64.9 ± 128.8 µl at nadir point. Residual fluid was positively correlated with baseline SRF (r = 0.76, p < 0.0001) and larger pigment epithelium detachment (r = 0.65, p = 0.0001). Polypoidal choroidal vasculopathy was associated with larger residual fluid (p = 0.0013).ConclusionsIncomplete anti-VEGF responders in nAMD showed significant mean fluid resolution between injections, typically after 2 weeks. However, complete resolution was the exception, and the amount of residual fluid varied greatly. To understand the role of the unresponsive fluid, further studies are needed.

Noninvasive optoacoustic microangiography reveals dose and size dependency of radiation-induced deep tumor vasculature remodeling.

Tumor microvascular responses may provide a sensitive readout indicative of radiation therapy efficacy, its time course and dose dependencies. However, direct high-resolution observation and longitudinal monitoring of large-scale microvascular remodeling in deep tissues remained challenging with the conventional microscopy approaches.We report on a non-invasive longitudinal study of morphological and functional neovascular responses by means of scanning optoacoustic (ОА) microangiography. In vivo imaging of CT26 tumor response to a single irradiation at varying dose (6, 12, and 18 Gy) has been performed over ten days following treatment. Tumor oxygenation levels were further estimated using diffuse optical spectroscopy (DOS) with a contact fiber probe.OA revealed the formation of extended vascular structures on the whole tumor scale during its proliferation, whereas only short fragmented vascular regions were identified following irradiation. On the first day post treatment, a decrease in the density of small (capillary-sized) and medium-sized vessels was revealed, accompanied by an increase in their fragmentation. Larger vessels exhibited an increase in their density accompanied by a decline in the number of vascular segments. Short-lasting response has been observed after 6 and 12 Gy irradiations, whereas 18 Gy treatment resulted in prolonged responses, up to the tenth day after irradiation. DOS measurements further revealed a delayed increase of tumor oxygenation levels for 18 Gy irradiations, commencing on the sixth day post treatment. The ameliorated oxygenation is attributed to diminished oxygen consumption by inhibited tumor cells but not to the elevation of oxygen supply.This work is the first to demonstrate the differential (size-dependent) nature of vascular responses to radiation treatments at varying doses in vivo. The OA approach thus facilitates the study of radiation-induced vascular changes in an unperturbed in vivo environment while enabling deep tissue high-resolution observations at the whole tumor scale.

Gamma-interferon inducible lysosomal thiolreductase (GILT) effect on breast tumor microenvironment through regulating CXCR6/CXCL16 axis.

e15081 Background: Gamma-interferon Inducible Lysosomal Thiolreductase (GILT) is constitutively expressed in most antigens endocytosed by antigen presenting cells (APCs), and its function is to catalyze the reduction of disulfide (S-S) bonds in protein substrates. The cytokine CXCL16 is one of the only two known plasma membrane chemokines which induces chemotaxis of activated T cells and bone marrow plasma cells in tumor microenvironment. It contains a free end folded by two sulfur bonds and therefore could also be a zymolyte of GILT. Previous studies found that specific receptor of CXCL16, CXCR6, was significantly overexpressed in breast cancer tissues and metastatic axillary lymph nodes. We suppose whether CXCR6/CXCL16 axis is regulated by GILT and affect tumor microenvironment, thereby eliciting specific anti-tumor immune responses in breast cancer (BC). Methods: GILT expression in BC was evaluated using publicly available data from The Cancer Genome Atlas (TCGA). GILT gene was analyzed in UALCAN ( http://ualcan.path.uab.edu/analysis-prot.html ) . In vitro, Immunohistochemistry (IHC) was conducted to examine the location and relation of GILT and CXCR6. Gene Set Enrichment Analysis (GSEA) was performed to mine the biological pathways involved in BC related GILT regulatory network. The expression of GILT at protein and RNA levels were detected by Western Blot and RT-PCR assay. Overexpression and knockdown of GILT in BC cell lines was carried out to further analyzed the correlation between GILT and CXCL16/CXCR6. Results: TCGA database showed that GILT expression was increased in the stroma of BC compared with normal, and was correlated to shorter BC overall survival. GSEA suggested that the expression of GILT was associated with chemotactic factors. Pearson analysis and IHC showed GILT had a strong correlation with CXCL16/CXCR6 axis in the aspect of angiogenesis and immunity. qRT-PCR and Western Blot assay also revealed that GILT had high expression in BC. Besides, patients with high expression of GILT in IHC simultaneously showed high immunoreactive to macrophage markers, which was related to neovascularization and anti-tumor immune responses. Compared with the normal breast cell line MCF-10A, GILT protein had high expression in Hs578T and low expression in MDA-MB-231 cell line. GILT was overexpressed in MDA-MB-231 and knockdown in Hs578T. Result showed that high level GILT promoted the production of CXCL16/CXCR6,while GILT siRNA knockdown inhibited the production of CXCL16/CXCR6. Conclusions: GILT could catalyze CXCL16 in BC, function as a key mechanism to affect tumor microenvironment through CXCR6/CXCL16 pathway. GILT-activated CXCL16 levels showed a strong connection with unfavorable outcomes in BC, which could be a potential biomarker of prognosis and a novel therapeutic target.

Retinal Phenotyping of Ferrochelatase Mutant Mice Reveals Protoporphyrin Accumulation and Reduced Neovascular Response.

PurposeHeme depletion, through inhibition of ferrochelatase (FECH), blocks retinal and choroidal neovascularization. Both pharmacologic FECH inhibition and a partial loss-of-function Fech mutation (Fechm1Pas) are associated with decreased neovascularization. However, the ocular physiology of Fechm1Pas mice under basal conditions has not been characterized. Here, we aimed to characterize the retinal phenotype of Fechm1Pas mice.MethodsWe monitored retinal vasculature at postnatal day 17, 2 months, and 6 months in Fechm1Pas homozygotes, heterozygotes, and their wild-type littermates. We characterized Fech substrate protoporphyrin (PPIX) fluorescence in the eye (excitation = 403 nm, emission = 628 nm), retinal function by electroretinogram, visual acuity by optomotor reflex, and retinal morphology by optical coherence tomography and histology. We stained vasculature using isolectin B4 and fluorescein angiography. We determined endothelial sprouting of retinal and choroidal tissue ex vivo and bioenergetics of retinal punches using a Seahorse flux analyzer.ResultsFundi, retinal vasculature, venous width, and arterial tortuosity showed no aberrations. However, VEGF-induced retinal and choroidal sprouting was decreased in Fechm1Pas mutants. Homozygous Fechm1Pas mice had pronounced buildup of PPIX in the posterior eye with no damage to visual function, bioenergetics, and integrity of retinal layers.ConclusionsEven with a buildup of PPIX in the retinal vessels in Fechm1Pas homozygotes, the vasculature remains normal. Notably, stimulus-induced ex vivo angiogenesis was decreased in Fechm1Pas mutants, consistent with reduced pathologic angiogenesis seen previously in neovascular animal models. Our findings indicate that Fechm1Pas mice are a useful model for studying the effects of heme deficiency on neovascularization due to Fech blockade.

Characterization of Inflammatory and Fibrotic Aspects of Tissue Remodeling of Acellular Dermal Matrix in a Nonhuman Primate Model.

Background:Human acellular dermal matrices (hADMs) are applied in various soft tissue reconstructive surgeries as scaffolds to support tissue remodeling and regeneration. To evaluate the clinical efficacy of hADM implants, it is integral that the hADM does not induce a host chronic inflammatory response leading to fibrotic encapsulation of the implant. In this study, we characterized the inflammatory and fibrosis-related tissue remodeling response of 2 commercial hADM products (SimpliDerm and AlloDerm RTU) in a nonhuman primate model using histology and gene expression profiling.Methods:Eighteen African green monkeys with abdominal wall defects were applied to evaluate the performance of SimpliDerm and AlloDerm RTU implants (N = 3) at 2, 4, and 12-weeks post-implantation. Using histology and gene expression profiling, tissue responses such as implant integration, degradation, cell infiltration, immune response, neovascularization, and pro-fibrotic responses over time were evaluated.Results:SimpliDerm showed a lower initial inflammatory response and slower implant degradation rate than AlloDerm RTU evidenced by histomorphological analysis. These factors led to a more anti-inflammatory and pro-remodeling microenvironment within SimpliDerm, demonstrated by lower TNFα levels and lower expression levels of pro-fibrotic markers, and promoted tissue repair and regeneration by 3-months post-implantation.Conclusions:Overall, histology and gene expression profiling analyses shown in this study demonstrated an effective model for analyzing hADM performance in terms of host inflammatory and fibrotic response. Further studies are warranted to fully evaluate the utility of this novel hADM in the clinical setting and verify the prognosis of our pre-clinical analysis model.

Effects of the COVID-19 pandemic on neovascular age-related macular degeneration and response to delayed Anti-VEGF treatment

ObjectivesTo investigate the effects of the COVID-19 pandemic on the treatment course of neovascular age-related macular degeneration (nAMD) patients who received anti-VEGF injection therapy with real-life data.MethodsThis retrospective study consisted of 116 eyes of 106 patients. Ophthalmic examination, assessment of best-corrected visual acuity (BCVA), optical coherence tomography (OCT) findings and data of last two visits before restrictions (V-2 and V-1) and the first visit (V0) after the release of national lockdown and subsequent visits (V1 and Vlast) were recorded. The lockdown period was determined by the time interval between March 11 and June 1, 2020.Main resultsThe injection interval before V-1 was significantly longer than the interval after V0 (2.56 ± 0.9 vs. 2.14 ± 0.8 months, P = 0.02). While the median central macular thickness (CMT) was significantly increased at V0 compared to V-1 [274(132–711) vs. 238(136–628), P < 0.001], the median CMT was significantly lower at V1 compared to V0 [256 (136–591) vs. 274(132–711), P = 0.003]. The median BCVA was 0.67(0.1–1.1) logMAR at V-1 and significantly worsened to 0.78 (0.1–1.2) logMAR at V0 (P = 0.003). Although the median BCVA improved to 0.69 logMAR (0.1–1.2) at Vlast, the difference did not reach statistical significance compared to V0 (P = 0.08).ConclusionTreatment delay due to the COVID-19 pandemic cause progression of nAMD and visual impairment. To plan more frequent anti-VEGF treatments and visits may be an appropriate approach until the disease stabilizes. However, it should be kept in mind that despite the improvement in OCT findings, the desired success in VA could not be achieved in the short term.

Flexor Tendon Injury and Repair. The Influence of Synovial Environment on the Early Healing Response in a Canine Model.

Environmental conditions strongly influence the healing capacity of connective tissues. Well-vascularized extrasynovial tendons typically undergo a robust wound-healing process following transection and repair. In contrast, avascular intrasynovial tendons do not mount an effective repair response. The current study tests the hypothesis that flexor tendons, as a function of their synovial environment, exhibit unique inflammatory, angiogenic, and metabolic responses to injury and repair. Flexor tendons present a distinct opportunity to test the study hypothesis, as they have proximal regions that are extrasynovial and distal regions that are intrasynovial. In an internally controlled study design, the second and fifth forepaw flexor tendons were transected and repaired in either the extrasynovial or the intrasynovial anatomical region. Histological, gene expression, and proteomics analyses were performed at 3 and 7 days to define the early biological events that drive synovial environment-dependent healing responses. Uninjured intrasynovial tendons were avascular, contained high levels of proteoglycans, and expressed inflammatory factors, complement proteins, and glycolytic enzymes. In contrast, extrasynovial tendons were well vascularized, contained low levels of proteoglycans, and were enriched in inflammation inhibitors and oxidative phosphorylation enzymes. The response to injury and repair was markedly different between the 2 tendon regions. Extrasynovial tendons displayed a robust and rapid neovascularization response, increased expression levels of complement proteins, and an acute shift in metabolism to glycolysis, whereas intrasynovial tendons showed minimal vascularity and muted inflammatory and metabolic responses. The regional molecular profiles of intact and healing flexor tendons revealed extensive early differences in innate immune response, metabolism, vascularization, and expression of extracellular matrix as a function of the synovial environment. These differences reveal mechanisms through which extrasynovial tendons heal more effectively than do intrasynovial tendons. To improve outcomes after operative repair, future treatment strategies should promote features of extrasynovial healing, such as enhanced vascularization and modulation of the complement system and/or glucose metabolism.