Higher Vascular Density Research Articles

Platelet-Derived Growth Factor Subunit A Strengthens the Neurovascular Unit and Inhibits Retinal Vascular Regression Under Hyperoxic Conditions

Retinopathy of prematurity (ROP) is primarily caused by the exposure of preterm infants with underdeveloped blood vessels to high oxygen concentrations. This damages the astrocytes that promote normal vascular development, leading to avascularity, pathological neovascularization, and retinal detachment, and even blindness as the disease progresses. In this study, the aim was to investigate the differences in the characteristics of astrocytes and blood vessels between wild-type (WT) and genetically modified mice overexpressing platelet-derived growth factor subunit A (PDGF-A) in the retina immediately after high oxygen exposure, a protocol in the oxygen-induced retinopathy (OIR) model of ROP. Our results showed that PDGF-A mice exhibited an increased population of astrocytes and higher vascular density than WT mice and that PDGF-A strengthened the resistance to hyperoxic conditions. In the OIR model, PDGF-A mice had reduced avascular zone areas following hyperoxia exposure. Furthermore, immunostaining for NG2 and CD31 showed that pericytes tended to regress earlier than endothelial cells, particularly at the vessel edges in both WT and transgenic mice, indicating relatively higher susceptibility to hyperoxia-induced damage. These findings suggest that PDGF-A plays a crucial role in stabilizing retinal vessels and may serve as a novel therapeutic target for ROP, highlighting the potential significance of PDGF-A in the pathological mechanisms of retinal diseases.

Exendin-4 promotes ischemia-reperfusion flap survival by upregulating Gpx4 to inhibit ferroptosis

BackgroundEffective drugs for preventing or treating skin flap necrosis remain elusive. In this study, we investigated the potential protective effect of exendin-4 against skin flap ischemia-reperfusion injury (IRI) through the inhibition of ferroptosis. MethodA rat abdomen was constructed with an island skin flap, and the superficial vascular pedicle of the abdominal wall was closed using a vascular clamp, which was removed after 8 h. Before surgery, RSL3 and ferrostatin-1 solutions were intraperitoneally injected. After the surgery, subcutaneous injections of exendin-4 were administered daily. The number of inflammatory cells, mean vascular density, collagen fiber content, and apoptosis and ferroptosis indicators were quantified 24 h after reperfusion. Survival, contraction rate, and blood perfusion of the skin flap were evaluated on days 1, 3, 5, and 7 after reperfusion. ResultsThe flap survival rate was significantly higher in the exendin-4 group than that in the injury group, whereas the contraction rate was lower. Compared with the injury group, the exendin-4 group showed less inflammatory cell infiltration, higher vascular density, and less collagen fiber loss. At the molecular level, the exendin-4 group demonstrated opposite or elevated expression of apoptosis and ferroptosis indicators than those in the injury group, with significantly increased glutathione peroxidase 4 (Gpx4). Ferroptosis inhibitors and agonists enhanced and reversed the protective effects of exendin-4, respectively. ConclusionExendin-4 alleviates skin flap IRI by upregulating Gpx4 expression to inhibit ferroptosis. Therefore, exendin-4 may serve as a novel clinical treatment for skin flap IRI.

Neurovascular and immune factors of vulnerability of substantia nigra dopaminergic neurons in non-human primates

Dopaminergic neurons in the ventral tier of the substantia nigra pars compacta (SNc) degenerate prominently in Parkinson’s disease (PD), while those in the dorsal tier and ventral tegmental area are relatively spared. The factors determining why these neurons are more vulnerable than others are still unrevealed. Neuroinflammation and immune cell infiltration have been demonstrated to be a key feature of neurodegeneration in PD. However, the link between selective dopaminergic neuron vulnerability, glial and immune cell response, and vascularization and their interactions has not been deciphered. We aimed to investigate the contribution of glial cell activation and immune cell infiltration in the selective vulnerability of ventral dopaminergic neurons within the midbrain in a non-human primate model of PD. Structural characteristics of the vasculature within specific regions of the midbrain were also evaluated. Parkinsonian monkeys exhibited significant microglial and astroglial activation in the whole midbrain, but no major sub-regional differences were observed. Remarkably, the ventral substantia nigra was found to be typically more vascularized compared to other regions. This feature might play some role in making this region more susceptible to immune cell infiltration under pathological conditions, as greater infiltration of both T- and B- lymphocytes was observed in parkinsonian monkeys. Higher vascular density within the ventral region of the SNc may be a relevant factor for differential vulnerability of dopaminergic neurons in the midbrain. The increased infiltration of T- and B- cells in this region, alongside other molecules or toxins, may also contribute to the susceptibility of dopaminergic neurons in PD.

Vascular regional analysis unveils differential responses to anti-angiogenic therapy in pancreatic xenografts through macroscopic photoacoustic imaging.

Pancreatic cancer (PC) is a highly lethal malignancy and the third leading cause of cancer deaths in the U.S. Despite major innovations in imaging technologies, there are limited surrogate radiographic indicators to aid in therapy planning and monitoring. Amongst the various imaging techniques Ultrasound-guided photoacoustic imaging (US-PAI) is a promising modality based on endogenous blood (hemoglobin) and blood oxygen saturation (StO 2 ) contrast to monitor response to anti-angiogenic therapies. Adaptation of US-PAI to the clinical realm requires macroscopic configurations for adequate depth visualization, illuminating the need for surrogate radiographic markers, including the tumoral microvessel density (MVD). In this work, subcutaneous xenografts with PC cell lines AsPC-1 and MIA-PaCa-2 were used to investigate the effects of receptor tyrosine kinase inhibitor (sunitinib) treatment on MVD and StO 2 . Through histological correlation, we have shown that regions of high and low vascular density (HVD and LVD) can be identified through frequency domain filtering of macroscopic PA images which could not be garnered from purely global analysis. We utilized vascular regional analysis (VRA) of treatment-induced StO 2 and total hemoglobin (HbT) changes. VRA as a tool to monitor treatment response allowed us to identify potential timepoints of vascular remodeling, highlighting its ability to provide insights into the TME not only for sunitinib treatment but also other anti-angiogenic therapies.

Theoretical modeling of tumor angiogenesis: the opposing effects of vascular endothelial growth factor and angiopoietin-2

Tumor microvascular networks are highly unstable, disorganized structures that obstruct cancer therapeutics by poorly distributing blood flow and oxygen. The elevated release of vascular endothelial growth factor (VEGF) by solid tumors has been widely implicated in pathological angiogenesis, or the growth of new blood vessels. However, how VEGF overexpression leads to the malformation of vascular networks is incompletely understood. Angiopoietin-2 (Ang2) is a destabilization factor that has been shown to promote sprouting in the presence of VEGF with hypoxia, but regression in its absence. Here, we hypothesize that the tumor environment, which sustains VEGF production under conditions of normoxia, also promotes Ang2-induced regression. A theoretical model is developed representing the 3-dimensional growth, remodeling, and pruning of microvascular networks in early-stage tumors to investigate the combined effects of VEGF and Ang2. Oxygen transport by the growing vascular network is simulated. VEGF production is induced by hypoxia and also elevated independent of oxygen levels in the expanding tumor region. Angiogenesis is stimulated in response to elevated VEGF levels. The model predicts that the release of VEGF in normoxic regions without counteracting factors results in very high tumor vascular densities compared to normal tissue, which is not observed experimentally. Introducing the regressive effects of Ang2 limits vascular density and produces greater variability in oxygen levels in the tumor region. Vascular networks in the tumor generated from the model show comparable densities to surrounding tissue, in which an elevated rate of sprouting is balanced by Ang2-induced regression. In this state, continuous turnover of vessels is predicted, while a relatively constant vascular density is maintained. These simulations suggest that the destabilizing role of Ang2 is an important factor that should be included in theoretical models for tumor angiogenesis. While Ang2 activity is already targeted in a number of clinical trials, clarifying the dynamic role of Ang2 may help advance angiogenesis-based therapeutic approaches. Supported by NIH grant HL034555. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Deferoxamine topical cream superior to patch in rescuing radiation-induced fibrosis of unwounded and wounded skin.

Topical patch delivery of deferoxamine (DFO) has been studied as a treatment for this fibrotic transformation in irradiated tissue. Efficacy of a novel cream formulation of DFO was studied as a RIF therapeutic in unwounded and excisionally wounded irradiated skin. C57BL/6J mice underwent 30 Gy of radiation to the dorsum followed by 4 weeks of recovery. In a first experiment, mice were separated into six conditions: DFO 50 mg cream (D50), DFO 100 mg cream (D100), soluble DFO injections (DI), DFO 1 mg patch (DP), control cream (Vehicle), and irradiated untreated skin (IR). In a second experiment, excisional wounds were created on the irradiated dorsum of mice and then divided into four treatment groups: DFO 100 mg Cream (W-D100), DFO 1 mg patch (W-DP), control cream (W-Vehicle), and irradiated untreated wounds (W-IR). Laser Doppler perfusion scans, biomechanical testing, and histological analysis were performed. In irradiated skin, D100 improved perfusion compared to D50 or DP. Both D100 and DP enhanced dermal characteristics, including thickness, collagen density and 8-isoprostane staining compared to untreated irradiated skin. D100 outperformed DP in CD31 staining, indicating higher vascular density. Extracellular matrix features of D100 and DP resembled normal skin more closely than DI or control. In radiated excisional wounds, D100 facilitated faster wound healing and increased perfusion compared to DP. The 100 mg DFO cream formulation rescued RIF of unwounded irradiated skin and improved excisional wound healing in murine skin relative to patch delivery of DFO.

Danger Zones of the Gluteal Anatomy: Improving the Safety Profile of the Gluteal Fat Grafting

IntroductionKnowledge of the vascular anatomy is critical to performing safe gluteal surgery. To date, only the course of the main blood vessels within the muscles has been outlined. These findings are based on MRI and CTA images that do not conform to a topographically standardized and normalized probability distribution.ObjectivesThe aim of this study was to develop a three-dimensional mapping of the gluteal zones of high vascular density in relation to anatomical landmarks.Materials and MethodsThis single-center retrospective cohort analysis comprised all consecutive patients who underwent cone-beam computed tomography (CBCT) scans between January 2016 and October 2021. The location of blood vessels in the gluteal region was allometrically normalized in relation to anatomical landmarks. Moreover, the caliber and area of the blood vessels were assessed.ResultsCBCT scans of 32 patients with an average age of 64 ± 12 years (range 34–87 years) were included. Fifty-three percent were female. The median [IQR] caliber of the intramuscular gluteal vessels was 1.47 [1.15–1.88] mm, significantly greater than that of the subcutaneous vessels 1.09 [0.72–1.44] mm (p < 0.001). Vascular density was higher intramuscularly, as 4.5% of the area of the muscle was occupied by blood vessels, as opposed to 0.3% in the adipose tissue.ConclusionThe analysis of the CBCT scans showed a higher vascular density and larger vessels intramuscularly. We, therefore, recommend the injection of autologous fat merely to the subcutaneous plane.Level of Evidence II This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Retinal Microvasculature Causally Affects the Brain Cortical Structure: A Mendelian Randomization Study

PurposeTo reveal the causality between retinal vascular density (VD), fractal dimension (FD) and brain cortex structure using Mendelian randomization (MR). DesignCross-sectional study. ParticipantsGenome-wide association studies of VD and FD involving 54,813 participants from the UK Biobank were used. The brain cortical features, including the cortical thickness (TH) and surficial area (SA), were extracted from 51,665 patients across 60 cohorts. SA and TH were measured globally and in 34 functional regions using magnetic resonance imaging. MethodsBidirectional univariable MR (UVMR) was used to detect the causality between FD, VD and brain cortex structure. Multivariable MR (MVMR) was used to adjust for confounding factors, including body mass index and blood pressure. Main Outcome MeasuresThe global and regional measurements of brain cortical surface area and cortical thickness. ResultsAt the global level, higher VD is related to decreased TH (β =-0.0140 mm, 95% CI: -0.0269 mm to -0.0011 mm, P = 0.0339). At the functional level, retinal FD is related to the TH of banks of the superior temporal sulcus and transverse temporal region without global weighted, as well as the SA of the posterior cingulate after adjustment. VD is correlated with the SA of subregions of the frontal lobe and temporal lobe, in addition to the TH of the inferior temporal, entorhinal and pars opercularis regions in both UVMR and MVMR. Bidirectional MR studies showed a causation between the SA of the parahippocampal and cauda middle frontal gyrus and retinal VD. No pleiotropy was detected. ConclusionsFD and VD causally influence the cortical structure and vice versa, indicating that the retinal microvasculature may serve as a biomarker for cortex structural changes. Our study provides insights into utilizing noninvasive fundus images to predict cortical structural deteriorations and neuropsychiatric disorders.

Abstract 13022: Myocardial Restoration and Functional Recovery Through Transplantation of Vascularized Cardiac Microtissues Derived From Human Induced Pluripotent Stem Cells in a Rat Model of Myocardial Infarction

Introduction: Cardiac regenerative therapy is acknowledged as a promising strategy for treating individuals suffering from severe heart failure. To address the current constraints of stem cell therapy, which primarily relies on indirect paracrine effects for functional improvement, the ideal goal would be to restore the damaged myocardium in the diseased heart. To achieve this, the transplantation of biomimetic heart tissues that closely resemble the natural structure of the heart is highly anticipated. Methods: We successfully generated 3-dimensional vascularized cardiac microtissues (VCMs) using human induced pluripotent stem cells (hiPSCs) through dynamic culture on thin cardiac tissue sheets (CTSs) composed of hiPSC-derived cardiomyocytes and vascular cells. These VCMs exhibited a robust vascular network comprising human vascular cells. Subsequently, we transplanted CTSs (n=6) or VCMs (n=6) onto an athymic rat model of myocardial infarction (MI), while a sham operation was performed on another group (n=6). To assess cardiac function, we employed cardiac magnetic resonance imaging. The survival of grafts, extension of fibrotic scars, neovascularization in the MI border zone, and the formation of a human vascular network within the engrafted region were evaluated through histopathology and light sheet fluorescence microscopy with tissue clearing techniques. Results: Functional analyses demonstrated that the VCM group exhibited the highest left ventricular ejection fraction (Sham: 36.5±0.9%, CTS: 43.5±2.9%, VCM: 55.2±2.5%, p &lt; 0.01). Histopathological evaluation revealed that the VCM group had a larger engraftment area, along with the smallest scar extension and the highest vascular density in the myocardial infarction border zone. Furthermore, LSFM revealed the presence of vascular networks originating from human cells within the engrafted VCMs. Conclusions: The transplantation of hiPSC-derived VCMs demonstrated remarkable therapeutic potential in a rat model of MI, leading to the formation of a vascular network by human cells. This approach might hold great promise as an effective strategy for efficiently restoring the damaged myocardium in hearts afflicted with disease.

Preliminary study of some of the main intratumor stroma components in gastric carcinomas.

The relationship between stromal compartment and tumor behavior in gastric carcinomas is still poorly understood and defined. Therefore, the authors started, with this preliminary study, an analysis of stromal compartment morphology and behavior in tumors arising from gastric mucosa epithelium. The study group included 75 patients operated for gastric carcinoma. Five parameters describing tumor morphology and behavior and eight parameters describing tumor stroma (TS) morphology were assessed. Histopathological examination included six serial sections of formalin-fixed, paraffin-embedded tumor tissue samples, stained with three classical stains and three antibodies to reveal the different parameters. For data comparison, Pearson's correlation test and the chi-squared (χ²) correlation test were used. Studied tumors were, usually, infiltrating, undifferentiated∕diffuse type, invasive in subserous spaces and with a Ki67 index higher than 20%. Collagen fibers dominated the stromal components, with a predominance of mature type and an average fibrillary index of 2.7. The whole amount of stromal components accounted for around one quarter of the tumor area. Mature collagen fibers were in opposite correlation with their immature counterpart, and both were in opposite correlation with smooth muscle fibers and expressed an opposite trend of correlation with components of vascular compartment. The whole amount of stromal components had divergent behavior with the components of vascular compartment. The latter expressed generally an opposite trend of correlation with individual fibrillary stromal components. We found only isolated relationships statistically significant between stromal components and tumor characteristics. TS is in a continuous remodeling process in relation to the evolution of tumor parenchyma, tumors less differentiated proving to have an immature stroma, with newly formed collagen fibers and higher vascular density. Further studies are required.

Melatonin Enhances the Viability of Random-Pattern Skin Flaps by Activating the NRF2 pathway

IntroductionRandom skin flap transplantation has been widely used in reconstructive and plastic surgery. As a well-known antioxidant, melatonin has the functions of eliminating reactive oxygen species (ROS), promoting angiogenesis, and protecting ischemia-reperfusion injury (IRI). We explored the effects of melatonin on random skin flap survival and the potential molecular mechanisms.Material and methodsA total of 72 rats were randomly assigned to the control group, the melatonin (MEL) group, and MEL + ML385 groups. After the construction of random skin flap model, these groups were treated with physiological saline, melatonin, and melatonin + ML385. The general conditions of random skin flaps were observed daily after the procedure. Laser doppler blood flow imaging was used to evaluate the subcutaneous vascular network. On postoperative day 7, the animals were euthanized to obtain flap specimens. Hematoxylin-Eosin staining was used to evaluate the vessel density. Immunohistochemistry, immunofluorescence staining, and western blotting were used to evaluate the expression of proteins involved in angiogenesis, oxidative stress, and inflammation.ResultsCompared to the control group, the MEL group exhibited lower tissue water, more abundant vascular, and higher vascular density, thereby enhancing the survival of random flaps. Additionally, the MEL group showed increased expression of angiogenesis-related proteins, enhanced expression of antioxidant proteins, and decreased expression of inflammatory factors. Furthermore, ML385, reversed the beneficial effect of melatonin on random skin flaps.ConclusionsThese findings of our present study demonstrated that melatonin promotes angiogenesis, inhibits oxidative stress, and inflammation by activating NRF2 signaling pathway, thus the improving the survival of random skin flaps.

Early Retinal Microvascular Changes Assessed with Swept-Source OCT Angiography in Type 1 Diabetes Patients without Retinopathy

Type 1 diabetes is a chronic disease that can lead to vision loss when diabetic retinopathy develops. Retinal microvascular alterations occur before the appearance of clinical signs on a fundus examination. This study aimed to analyze retinal vascular parameters on optical coherence tomography angiography (OCT-A) in patients with type 1 diabetes without diabetic retinopathy in comparison with non-diabetic volunteers. This cross-sectional study was conducted at Dijon University Hospital from 2018 to 2020. Vascular densities were measured using macular OCT-A. In total, 98 diabetes patients and 71 non-diabetic volunteers were enrolled. A statistically significant lower vascular density of the inner circle was found in the superficial capillary plexus (SCP) in the diabetes group (p < 0.01). There was a statistically significant correlation between central vascular density in the deep capillary plexus (DCP) and total daily insulin intake (p = 0.042); furthermore, use of the FreeStyle Libre (FSL) device was associated with higher vascular densities in both the SCP (p = 0.034 for outer circle density) and DCP (p < 0.01 for inner circle density and p = 0.023 for outer circle density). Retinal microvascularization was early-altered in type 1 diabetes, and using the FSL device seemed to preserve retinal microvascularization.

Una tumefazione della parete addominale

A 12-year-old girl complained of a painless, slightly pulsatile abdominal lump for two months. Radiological imaging showed a fusiform lesion within the abdominal wall, between the external and internal oblique muscle, with nonhomogeneous echo-structure and high vascular density at the colour-power-Doppler analysis. The findings suggested an intramuscular arteriovenous malformation. The patient underwent a two-step treatment, namely a preoperative transarterial angioembolization of the nourishing vessels through the right femoral artery that was followed by a surgical resection of the lesion. Recovery was uneventful and during the follow-up no relapses were reported.

Engineering pedicled vascularized bladder tissue for functional bladder defect repair.

An engineered bladder construct that mimics the structural and functional characteristics of native bladder is a promising therapeutic option for bladder substitution. We previously showed that pedicled vascularized smooth muscle tissue fabricated by grafting smooth muscle cell (SMC) sheets onto an axial capsule vascular bed had the potential for reliable bladder reconstruction. In this study, we investigated the feasibility of buccal mucosa graft (BMG) integration with the pedicled vascularized smooth muscle tissue to generate a full-layer pedicled vascularized bladder construct. BMG transplanted onto vascularized smooth muscle tissue showed good survival and developed into a pedicled vascularized bladder construct with full-layer structures, appropriate thickness, abundant vascularization, and effective barrier function. Then the full-thickness bladder defects were, respectively, reconstructed by pedicled capsule tissue (pedicled capsule group), nonpedicled vascularized bladder construct (nonpedicled construct group), and pedicled vascularized bladder construct (pedicled construct group). The picrosirius red (PSR) staining and immunohistochemistry results showed minimal fibrosis, maximal smooth muscle proportion, and high vascular density in the pedicled construct group. A continuous mucosal layer was observed only in the pedicled construct group. Moreover, morphological and functional studies revealed better bladder compliance and good ductility in the pedicled construct group. Overall, these results suggested that the BMG could be well integrated with vascularized smooth muscle tissue and generated a pedicled, fully vascularized, and structurally organized bladder construct, which facilitated structural remodeling and functional recovery and could become an alternative to bladder reconstruction.

Digital Staging of Hepatic Hemangiomas Reveals Spatial Heterogeneity in Endothelial Cell Composition and Vascular Senescence.

Hepatic hemangioma (HH) is the most common benign primary liver tumor; however, despite its high prevalence, a stage-specific classification of this tumor is currently missing. For a spatial stage-specific classification, a tissue microarray (TMA) consisting of 98 HHs and 80 hemangioma margins and 78 distant liver tissues was digitally analyzed for the expression of 16 functional and vascular niche-specific markers. For cross-correlation of histopathology and functional characteristics, computed tomography/MRI imaging data of 28 patients were analyzed. Functional and morphological analyses revealed a high level of intra- and interpatient heterogeneity, and morphological heterogeneity was observed with regard to cellularity, vascular diameter, and endothelial cell subtype composition. While regressed hemangiomas were characterized by low blood vessel density, low beta-catenin levels, and a microvascular phenotype, non-regressed HHs showed a pronounced cellular and architectural heterogeneity. Functionally, cellular senescence-associated p16 expression identified an HH subgroup with high vascular density and increased lymphatic endothelial cell content. Histological HH regions may be grouped into spatially defined morphological compartments that may reflect the current region-specific disease stage. The stage-specific classification of HHs with signs of regression and vascular senescence may allow a better disease course-based and cell state-based subtyping of these benign vascular lesions.

Normative Data and Determinants of Macular, Disc, and Peripapillary Vascular Density in Healthy Myopic Children Using Optical Coherence Tomography Angiography.

ObjectiveTo establish a normative database for the vascular density (VD) in macular, disc, and peripapillary regions in healthy myopic children and to evaluate associated ocular features with optical coherence tomography angiography (OCTA).MethodsThis was an observational, prospective and cross-sectional study. 776 Chinese healthy myopic children (375 boys and 401 girls) were enrolled, mean (±SD) age 9.84 ± 1.98 (range 6–16) years. En-face angiogram OCTA was performed on 6 mm × 6 mm retinal and 4.5 mm × 4.5 mm disc regions. VD measurements in the macular retina were segmented into the four regions: superficial capillary plexus (SCP), intermediate capillary plexus (ICP), deep capillary plexus (DCP), and choriocapillaris (CC). Correlations between macular, disc, and peripapillary VD and possible influencing factors [age, gender, axial length (AL), spherical equivalent refraction (SER), right/left eye, and signal strength index (SSI)] were assessed by Pearson’s correlation and multivariate regression analysis.ResultsFor macular scans, the corrected VD in the ICP region was (48.25 ± 4.24)% for the whole macular retina. The macular ICP VD in most sections was lower than the SCP, DCP, and CC (all P < 0.001). The corrected VD in CC was (72.96 ± 4.42)% for the whole macular retina. The macular CC VD in every section was all higher than the SCP, ICP, and DCP (all P < 0.001). The size of foveal avascular zone (FAZ) and foveal VD 300 (FD-300) was 0.28 mm2± 0.10 mm2 and (58.43 ± 4.17)% respectively. For disc scans, the corrected VD was (58.04 ± 2.73)% for the whole disc area. Both AL and SER were strongly correlated with ICP, DCP, and CC VD in all regions (all P < 0.01). Larger SSI was correlated with a lower VD in the SCP and ICP, and a higher VD in DCP and CC (all P < 0.01).ConclusionVascular density values provide large scale normative data on macular, disc, and peripapillary vascular parameters in a large sample of healthy myopic children with OCTA measured in the four different retinal plexuses and regions. The VD in different regions had various influencing factors; mainly a close correlation with AL and SSI.

Abstract LB057: Bevacizumab leaves a photoacoustic fingerprint in breast cancer mouse models

Abstract Background: Photoacoustic tomography (PAT) provides a direct readout of tumor haemoglobin (Hb) concentration and oxygenation. This readout could be used to provide an early indication of response to anti-angiogenic drugs, thus optimizing the management of these therapies. Experimental Procedures: Two cohorts of nude Balb/c were inoculated with either estrogen-dependent MCF-7 (n=7) or estrogen-independent MDA-MB-231 (n=19) cells. Mice were randomly split into Control (Ctrl) and Bevacizumab (Bev,10 mg/Kg, IP, weekly) groups. PAT was performed weekly, starting just before enrolment. Oxy- and deoxy-Hb were quantified in the tumour and reference areas. Total Hb (THb) and O2 saturation (SO2) were calculated. Blood samples and tissues were collected after imaging. Hypoxia (Hypoxyprobe and CA-IX) and vascular density/maturity (CD31 and ASMA) were analyzed by immunohistochemistry. Circulating levels of human and mouse vascular endothelial growth factor (hVEGF and mVEGF) and Hb were measured. Summary of New Data: Reflecting clinical observations, the MCF-7 Bev group and most of the mice from the MDA-MB-231 Bev group (8/11) showed no improvement in survival with Bev treatment. All resistant tumours (Bev MCF-7 and Bev MDA-MB-231 non-responders (Bev-NR)) showed an increase in hVEGF production (Ctrl vs Bev; 73.0513.06 vs. 497103.9 for MCF-7; 247.984.81 vs. 330.4 85.97 for Bev NR). MDA-MB-231 responders (Bev-R) showed a significant decrease in hVEGF (247.984.81 vs. 80.53 26.13). These results indicate that VEGF was only successfully sequestered in a small subset of animals (3/11) and that VEGF overload might be a resistance mechanism. At the final time point, PAT showed no difference between Ctrl and Bev-NR in THb for either group (THbMDA-MB-231 7.2±1.3 vs. 5.5±1.2). THb was found significantly increased in the Bev-R group by PAT (13.9±3.3 p-valuevs.Crtl=0.0339; p-valuevs.Bev=0.0072) and biochemical measurements. SO2 showed a slight but significant decrease between Ctrl and Bev-NR (0.58±0.03 vs. 0.48±0.01). A dramatic decrease was seen in the Bev-R group (0.31±0.07) and significantly different from Ctrl and Bev-NR. SO2 was sensitive to early changes, SO2 values increased in Ctrl and Bev-NR 48h after enrolment (p-values paired t-test pCtrl= 0.0134; pNR=0.0268) while Bev-R shows a trend (p=0.0649). Analysis of SO2MSOT over time shows a significant (p&amp;lt;0.0001) change in slope at 3 weeks after enrolment in the Bev-R group compared to either of the Ctrl or Bev-NR groups. Our results indicate that PAT SO2 can differentiate responders at very early stages of the treatment. Histologically, we observed fewer blood vessels but better structured in Bev-NR than in Bev-R, which showed higher vascular density and also higher levels of hypoxia makers. Statement of the Conclusions: Our results postulates PAT as a low-cost, label-free and non-invasive candidate to monitor response to Bevacizumab over time. Our results also indicate that tumours with functional vasculature may be resistant to Bevacizumab treatment. Citation Format: Isabel Quiros-Gonzalez, Michal R. Tomaszewski, Monika A. Golinska, Emma Brown, Laura Ansel-Bollepalli, Lina Hacker, Dominique-Laurent Couturier, Rosa M. Sainz, Sarah E. Bohndiek. Bevacizumab leaves a photoacoustic fingerprint in breast cancer mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB057.

Validation of a Novel Three-Dimensional (3D Fusion) Gross Sampling Protocol for Clear Cell Renal Cell Carcinoma to Overcome Intratumoral Heterogeneity: The Meet-Uro 18 Study.

We aimed to overcome intratumoral heterogeneity in clear cell renal cell carcinoma (clearRCC). One hundred cases of clearRCC were sampled. First, usual standard sampling was applied (1 block/cm of tumor); second, the whole tumor was sampled, and 0.6 mm cores were taken from each block to construct a tissue microarray; third, the residual tissue, mapped by taking pieces 0.5 × 0.5 cm, reconstructed the entire tumor mass. Precisely, six randomly derived pieces of tissues were placed in each cassette, with the number of cassettes being based on the diameter of the tumor (called multisite 3D fusion). Angiogenic and immune markers were tested. Routine 5231 tissue blocks were obtained. Multisite 3D fusion sections showed pattern A, homogeneous high vascular density (10%), pattern B, homogeneous low vascular density (8%) and pattern C, heterogeneous angiogenic signatures (82%). PD-L1 expression was seen as diffuse (7%), low (33%) and absent (60%). Tumor-infiltrating CD8 scored high in 25% (pattern hot), low in 65% (pattern weak) and zero in 10% of cases (pattern desert). Grading was upgraded in 26% of cases (G3–G4), necrosis and sarcomatoid/rhabdoid characters were observed in, respectively, 11 and 7% of cases after 3D fusion (p = 0.03). CD8 and PD-L1 immune expressions were higher in the undifferentiated G4/rhabdoid/sarcomatoid clearRCC subtypes (p = 0.03). Again, 22% of cases were set to intermediate to high risk of clinical recurrence due to new morphological findings of all aggressive G4, sarcomatoid/rhabdoid features by using 3D fusion compared to standard methods (p = 0.04). In conclusion, we propose an easy-to-apply multisite 3D fusion sampling that negates bias due to tumor heterogeneity.

\u03b2IV-spectrin as a stalk cell-intrinsic regulator of VEGF signaling

Defective angiogenesis underlies over 50 malignant, ischemic and inflammatory disorders yet long-term therapeutic applications inevitably fail, thus highlighting the need for greater understanding of the vast crosstalk and compensatory mechanisms. Based on proteomic profiling of angiogenic endothelial components, here we report βIV-spectrin, a non-erythrocytic cytoskeletal protein, as a critical regulator of sprouting angiogenesis. Early loss of endothelial-specific βIV-spectrin promotes embryonic lethality in mice due to hypervascularization and hemorrhagic defects whereas neonatal depletion yields higher vascular density and tip cell populations in developing retina. During sprouting, βIV-spectrin expresses in stalk cells to inhibit their tip cell potential by enhancing VEGFR2 turnover in a manner independent of most cell-fate determining mechanisms. Rather, βIV-spectrin recruits CaMKII to the plasma membrane to directly phosphorylate VEGFR2 at Ser984, a previously undefined phosphoregulatory site that strongly induces VEGFR2 internalization and degradation. These findings support a distinct spectrin-based mechanism of tip-stalk cell specification during vascular development.

Therapeutic potential of clinical-grade human induced pluripotent stem cell-derived cardiac tissues

ObjectivesTo establish a protocol to prepare and transplant clinical-grade human induced pluripotent stem cell (hiPSC)-derived cardiac tissues (HiCTs) and to evaluate the therapeutic potential in an animal myocardial infarction (MI) model. MethodsWe simultaneously differentiated clinical-grade hiPSCs into cardiovascular cell lineages with or without the administration of canonical Wnt inhibitors, generated 5- layer cell sheets with insertion of gelatin hydrogel microspheres (GHMs) (HiCTs), and transplanted them onto an athymic rat MI model. Cardiac function was evaluated by echocardiography and cardiac magnetic resonance imaging and compared with that in animals with sham and transplantation of 5-layer cell sheets without GHMs. Graft survival, ventricular remodeling, and neovascularization were evaluated histopathologically. ResultsThe administration of Wnt inhibitors significantly promoted cardiomyocyte (CM) (P < .0001) and vascular endothelial cell (EC) (P = .006) induction, which resulted in cellular components of 52.0 ± 6.1% CMs and 9.9 ± 3.0% ECs. Functional analyses revealed the significantly lowest left ventricular end-diastolic volume and highest ejection fraction in the HiCT group. Histopathologic evaluation revealed that the HiCT group had a significantly larger median engrafted area (4 weeks, GHM(-) vs HiCT: 0.4 [range, 0.2-0.7] mm2 vs 2.2 [range, 1.8-3.1] mm2; P = .005; 12 weeks, 0 [range, 0-0.2] mm2 vs 1.9 [range, 0.1-3.2] mm2; P = .026), accompanied by the smallest scar area and highest vascular density at the MI border zone. ConclusionsTransplantation of HiCTs generated from clinical-grade hiPSCs exhibited a prominent therapeutic potential in a rat MI model and may provide a promising therapeutic strategy in cardiac regenerative medicine.