Capillary Sprouts Research Articles

During Postnatal Ontogenesis, the Development of a Microvascular Bed in an Intestinal Villus Depends on Intussusceptive Angiogenesis.

The mechanisms responsible for the growth and development of vascular beds in intestinal villi during postnatal ontogenesis remain enigmatic. For instance, according to the current consensus, in the sprouting type of angiogenesis, there is no blood flow in the rising capillary sprout. However, it is known that biomechanical forces resulting from blood flow play a key role in these processes. Here, we present evidence for the existence of the intussusception type of angiogenesis during the postnatal development of micro-vessel patterns in the intestinal villi of rats. This process is based on the high-level flattening of blood capillaries on the flat surfaces of intestinal villi, contacts among the opposite apical plasma membrane of endothelial cells in the area of inter-endothelial contacts, or the formation of bridges composed of blood leucocytes or local microthrombi. We identified factors that, in our opinion, ensure the splitting of the capillary lumen and the formation of two parallel vessels. These phenomena are in agreement with previously described features of intussusception angiogenesis.

Dentin matrix protein 1 and HUVEC-ECM scaffold promote the differentiation of human dental pulp stem cells into endothelial lineage: implications in regenerative medicine.

Reprograming of the dental pulp somatic cells to endothelial cells is an attractive strategy for generation of new blood vessels. For tissue regeneration, vascularization of engineered constructs is crucial to improve repair mechanisms. In this study, we show that dentin matrix protein 1 (DMP1) and HUVEC-ECM scaffold enhances the differentiation potential of dental pulp stem cells (DPSCs) to an endothelial phenotype. Our results show that the differentiated DPSCs expressed endothelial markers CD31 and VE-Cadherin (CD144) at 7 and 14days. Expression of CD31 and VE-Cadherin (CD144) were also confirmed by immunofluorescence. Furthermore, flow cytometry analysis revealed a steady increase in CD31 and VE-Cadherin (CD144) positive cells with DMP1 treatment when compared with control. In addition, integrins specific for endothelial cells were highly expressed during the differentiation process. The endothelial cell signature of differentiated DPSCs were additionally characterized for key endothelial cell markers using gene expression by RT-PCR, Western blotting, immunostaining, and RNA-seq analysis. Furthermore, the angiogenic phenotype was confirmed by tubule and capillary sprout formation. Overall, stimulation of DPSCs by DMP1 and use of HUVEC-ECM scaffold promoted their differentiation into phenotypically, transcriptionally, and functionally differentiated bonafide endothelial cells. This study is novel, physiologically relevant and different from conventional strategies.

Exploring the Potential of Skin Cell Culture and the Vital Role of Skin Grafts in Regenerative Medicine and Biology

The skin is acknowledged as the biggest organ, covering the exterior of the body and serving as a physical barrier. The integumentary system is made up of three layers of tissue: the epidermis, dermis, and hypodermis. For transplantation, epidermal cell cultures have been around for a while. The epidermal stem cells can proliferate and self-renew in a manner that allows for rapid healing since they are unipotent, meaning they can only produce one type of cell. In therapeutic terms, cultured keratinocytes have been described as an autologous cell-based therapy that provides a source of autologous tissue, reducing the need for large donor sites and minimizing the risk of immune rejection. This review explores the composition and functioning of the skin’s extracellular matrix, along with strategies to incorporate ECM components into the cell culture techniques. Rebuilding skin flaws without consideration for side effects is skin grafting. The tissue lacks its circulatory supply and must receive it from the bed in which it is inserted. The skin cells have contributed to developing skin graft and tissue engineering approaches for repairing damaged skin. Similar to grafting, the skin graft is divided into sections based on the quantity of dermis involved: split-thickness skin and full-thickness skin. Transplanting autologous keratinocytes is an alternative to autologous skin grafting. In the wound bed, epidermal stem cells encourage angiogenesis. After invading the fibrin wound clot, angiogenic capillary sprouts form a microvascular network throughout the granulation tissue in a few days. In this article, authors will review the current methods and application of skin cell culture for transplantation including different cell sources, culture conditions, and transplantation techniques. The future direction of this approach is in the context of regenerative medicine and tissue engineering.

Time-Lapse Observation of Cell Dynamics During Angiogenesis Using the Rat Mesentery Culture Model.

The ability to track cells and their interactions with other cells during physiological processes offers a powerful tool for scientific discovery. An ex vivo model that enables real-time investigation of cell migration during angiogenesis in adult microvascular networks would enable observation of endothelial cell dynamics during capillary sprouting. Angiogenesis is defined as the growth of new blood vessels from existing ones and involves multiple cell types including endothelial cells, pericytes, and interstitial cells. The incorporation of these cell types in a physiologically relevant environment, however, represents a challenge for biomimetic model development. Recently, our laboratory has developed the rat mesentery culture model, which enables investigation of angiogenesis in an intact tissue. The objective of this chapter is to detail a protocol for tracking cellular dynamics during angiogenesis using the rat mesentery tissue culture model. The method involves harvesting mesentery tissues from adult SD-EGFP rats, culturing them in MEM+10% fetal bovine serum, and imaging network regions over the time course of angiogenesis. In example applications, time-lapse comparison of microvascular networks in cultured tissues confirmed dramatic increases in GFP-positive capillary sprouting and GFP-positive segment density. Additionally, tracking of individual capillary sprout extensions revealed their ability to "jump" by disconnecting from one vessel segment and reconnecting to another segment in the network. GFP-positive sprouts were also capable of undergoing subsequent regression. The representative results support the use of the rat mesentery culture model for identifying and tracking cellular dynamics during angiogenesis in intact microvascular networks.

PDGF-BB signaling via PDGFR-β regulates the maturation of blood vessels generated upon vasculogenic differentiation of dental pulp stem cells.

A functional vascular network requires that blood vessels are invested by mural cells. We have shown that dental pulp stem cells (DPSC) can undergo vasculogenic differentiation, and that the resulting vessels anastomize with the host vasculature and become functional (blood carrying) vessels. However, the mechanisms underlying the maturation of DPSC-derived blood vessels remains unclear. Here, we performed a series of studies to understand the process of mural cell investment of blood vessels generated upon vasculogenic differentiation of dental pulp stem cells. Primary human DPSC were co-cultured with primary human umbilical artery smooth muscle cells (HUASMC) in 3D gels in presence of vasculogenic differentiation medium. We observed DPSC capillary sprout formation and SMC recruitment, alignment and remodeling that resulted in complex vascular networks. While HUASMC enhanced the number of capillary sprouts and stabilized the capillary network when co-cultured with DPSC, HUASMC by themselves were unable to form capillary sprouts. In vivo, GFP transduced human DPSC seeded in biodegradable scaffolds and transplanted into immunodeficient mice generated functional human blood vessels invested with murine smooth muscle actin (SMA)-positive, GFP-negative cells. Inhibition of PDGFR-β signaling prevented the SMC investment of DPSC-derived capillary sprouts in vitro and of DPSC-derived blood vessels in vivo. In contrast, inhibition of Tie-2 signaling did not have a significant effect on the SMC recruitment in DPSC-derived vascular structures. Collectively, these results demonstrate that PDGF-BB signaling via PDGFR-β regulates the process of maturation (mural investment) of blood vessels generated upon vasculogenic differentiation of human dental pulp stem cells.

Estimation of shear stress values along endothelial tip cells past the lumen of capillary sprouts.

Shear stress is recognized as a regulator of angiogenesis. However, the shear stress experienced by the endothelial cells of capillary sprouts remains unknown. The objective of this study was to estimate shear stress due to local interstitial flow along endothelial tip cells at the end of the capillary sprout lumen. Computational fluid dynamics were used to model flow within a blind-ended vessel, transendothelial flow across the vessel wall, and flow within the surrounding perivascular/interstitial space. Shear stress along the wall of the tip cells was calculated while varying sprout length, perivascular space channel width, and vessel wall hydraulic conductivity. Increasing sprout length, increasing wall hydraulic conductivity, and decreasing perivascular space width increased shear stress magnitude. Wall shear stress magnitude within the lumen ranged from 0.015 to 0.55dyne/cm2 at the sprout entrance and linearly decreased to near zero at the base of the tip cells. Tip cell wall shear stress magnitude due to interstitial flow ranged from 0.009 to 4.65dyne/cm2. In 3 out of 8 cases, shear stress magnitude was above 1dyne/cm2 and considered physiologically relevant. The results provide a framework for discussing the role of local mechanical cues in regulating endothelial cell dynamics involved in angiogenesis. Mainly, interstitial flows may generate physiologically relevant shear stresses on tip cells in certain scenarios. This source of tip cell shear stress has not been previously considered or modeled.

Neonatal vitamin A administration increases intramuscular fat by promoting angiogenesis and preadipocyte formation

To explore the effects and underlying mechanism of vitamin A on beef marbling fat development, angus steers were injected vitamin A at birth and 1 month of age and in vitro experiments were performed to investigate the effects of retinoic acid (RA) on angiogenesis and adipogenesis of intramuscular stromal vascular (SVF) cells. Results showed that vitamin A administration increased the intramuscular PDGFRα+ adipose progenitors, improved adipogenic potential of intramuscular SVF cells and dramatically upregulated VEGFA. At slaughter, vitamin A increased intramuscular triacylglycerols by 45% without affecting overall fatness. In a 3D culture system, RA promoted capillary sprout development and promoted the subsequent adipogenesis of intramuscular SVF cells by activating VEGFA/VEGFR2 signaling. However, during terminal adipogenesis, RA downregulated PPARγ, C/EBPα and inhibited lipid accumulation. In conclusion, vitamin A/RA upregulate VEGFA and stimulate intramuscular vascular capillary development, which increases intramuscular adipose progenitors and contributes to adipocyte formation. When administrated at neonatal stage, vitamin A promotes beef marbling development without affecting overall fatness.

Chemotherapy, Microvascular Function, and Angiogenesis ‐ a Longitudinal Study

RationaleAnti‐cancer therapies (CTx), such as anthracyclines (e.g., doxorubicin, Dox), have known anti‐angiogenic effects in vitro, and inhibition of angiogenesis has known cardiotoxic effects. Cardiovascular (CV) toxicities that result from CTx significantly contribute to the morbidity and mortality of breast cancer survivors. While the negative impact of CTx, including Dox, on isolated cardiomyocytes is well‐established, significantly less is known about the effects of CTx on microvascular (MV) function and angiogenic potential in patients. Our goal was to understand how CTx damages the microcirculation in order to further the understanding of mechanisms that contribute to systemic cardiovascular toxicity. Utilizing a longitudinal study design of neoadjuvant breast cancer patients, adipose tissue was collected before, during, and after CTx treatment.MethodsAngiogenic potential was evaluated using a matrigel angiogenesis assay of patient adipose. Data are presented as a percentage of wells with capillary sprouts and area of capillary sprouting ± standard error. In addition, we investigated the expression of a subset of genes involved in angiogenesis regulation (initiation/sprouting, migration, ECM remodeling, maturation, and permeability). Freshly isolated microvessels were evaluated for dilation to flow (FMD) and acetylcholine (Ach). Cardiac function was monitored via standard clinical echocardiogram. Significance was determined using paired t‐test (p ≤ 0.05*).ResultsPercentage of wells with capillary sprouts was impaired in adipose samples collected during CTx (26.3% ± 13.6%, N=4) and one month post CTx (44.3% ± 9.1%*, N=9) compared to pre‐CTx (67.1% ± 4.7%*, N=13). However, sprouting area only changed during CTx (15.0 mm2± 8.5*, N=4) compared to pre‐CTx (68.3 mm2± 10.2, N=13). One month post CTx, the sprouting area (56.8 mm2± 19.1, N=9) was not significantly different than pre‐CTx. Supporting this change in angiogenesis function, we observe changes in expression of genes involved in initiation of angiogenesis during CTx (VEGFA, VEGFB, FLT1) that show recovery of expression post‐CTx. We also observe changes in expression of genes involved in maturation of angiogenesis post CTx (KLF4, VCAM1, MMP8). Some clinical signs of reduced cardiac function were observed post CTx after microvascular and angiogenic changes were observed.ConclusionOur data show pathological impairment in angiogenic potential and gene expression in adipose tissue of breast cancer patients in a longitudinal study. These data support our observed defects in MV function (response to FMD and Ach) during and after CTx treatment. We observed significant reductions in MV function and capillary sprouting during treatment with CTx in the absence of a clinically relevant reduction in cardiac function. Our findings suggest that CTx‐induced MV dysfunction precedes and may contribute to future adverse CV events.

A Novel ex vivo Method for Investigating Vascularization of Transplanted Islets

Revascularization of transplanted pancreatic islets is critical for survival and treatment of type 1 diabetes. Questions concerning how islets influence local microvascular networks and how networks form connections with islets remain understudied and motivate the need for new models that mimic the complexity of real tissue. Recently, our laboratory established the rat mesentery culture model as a tool to investigate cell dynamics involved in microvascular growth. An advantage is the ability to observe blood vessels, lymphatics, and immune cells. The objective of this study was to establish the rat mesentery tissue culture model as a useful tool to investigate islet tissue integration. DiI-labeled islets were seeded onto adult rat mesentery tissues and cultured for up to 3 days. Live lectin labeling enabled time-lapse observation of vessel growth. During culture, DiI-positive islets remained intact. Radial lectin-positive capillary sprouts with DiI labeling were observed to form from islets and connect to host networks. Lectin-positive vessels from host networks were also seen growing toward islets. PECAM and NG2 labeling confirmed that vessels sprouting from islets contained endothelial cells and pericytes. Our results introduce the rat mesentery culture model as a platform for investigating dynamics associated with the initial revascularization of transplanted islets.

Molecular Mechanism of Traditional Chinese Ointment of Xuzhou Qufu Shengji in Infected Wounds.

Background Xuzhou Qufu Shengji Ointment (QFSJO) has been used in hospital and private medication for more than 30 years to treat the infective wounds after trauma. However, molecular investigation is lacking. This study used rats to explore the healing mechanism of QFSJO in promoting wound healing in human. Methods One circular incision was individually generated on the back of 30 rats in three groups and challenged with 108 CFU (0.3 mL) of Staphylococcus aureus. Then, one of the trauma groups was treated with QFSJO gauze, and the control group was covered with a piece of Vaseline gauze, while the western medicine group was treated with erythromycin in a similar way. The dressing change of all the groups was performed once a day for three weeks. The anti-inflammation and proangiogenesis of QFSJO were evaluated by enzyme-linked immunosorbent assay (ELISA). The levels of angiogenesis associated factors, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF), hydroxyproline, and hemoglobin, were measured according to ELISA. The immunohistochemistry of CD31 and CD34 expression in granulation tissue was demonstrated and quantitatively analyzed for angiogenesis in granulation tissue in sites. Results A faster wound healing ratio was observed in QFSJO-dressing-treated group than Vaseline- and erythrocin-treated groups. ELISA results showed that QFSJO promoted VEGF and b-FGF levels significantly in early stage of wound healing. QFSJO dressing group also showed an enhanced hydroxyproline and hemoglobin in granulation tissue. The expressions of CD31 and CD34 in granulation tissue of QFSJO group were higher than in the Vaseline and erythrocin groups. Conclusion QFSJO improved the healing rate of the infective wounds by promoting the angiogenesis of granulation tissue and inhibiting the inflammation of the trauma tissue. Our finding suggests that QFSJO is able to help angiogenic capillary sprouts for collagen accumulates in the granulation tissue.

The notion of fractals in tumour angiogenic sprout initiation model based on cellular automata

Tumour growth is considered chaotic, poorly controlled growth as its cells and vasculatures are irregular in shape. The vascular system of tumour is a complex network and its architectural complexity and geometrical characteristics cannot be defined with Euclidian geometry as it is only capable of expressing regular or smooth objects whereas, fractal geometry describes an object with irregularities and it is therefore well suited to quantify those morphological characteristics. In this paper, we develop a cellular automata (CA) based discrete model that tries to mimic all the features and make useful predictions of tumour angiogenesis with computer-coded rules. From the interactions of several species in 2-D tissue space, we study the effects of these various biological factors on the formation of the capillary sprouts. The model also captures anastomoses and branching phenomena in capillary blood vessels. It is observed that chemotaxis and randomness are the essential components for the capillary sprout motility whereas, haptotaxis works as an add-on feature. The model is also validated with partial sensitivity analysis. The fractal properties of the growing capillary sprouts in the model are investigated. The characterization of these fractal objects (capillary sprouts) is provided by the box-counting method. The model shows, the morphological change in the early stages of the vascularization process where the capillary sprouts are formed from the surrounding blood vessels. The model successfully simulates the morphological aspects of capillary vessel growth during tumour angiogenesis. The estimated fractal dimension is very close to the previously measured range. This is established our hypothesis about the fractal nature of vascular network pattern and our CA-based discrete model for capillary sprout initiation during tumour angiogenesis.

Computational Evaluation of Wall Shear Stress Experienced by Endothelial Tip Cells along Capillary Sprouts

Shear stress is commonly implicated as a regulator of angiogenesis. However, critical questions remain unanswered. Do endothelial cells within a capillary sprout lumen experience shear stress? Can tip cells that extend past the sprout lumen experience shear stress? A full understanding of how shear stresses influence endothelial cell phenotype and function during angiogenesis requires the identification of local stress distribution along capillary sprouts. However, the actual shear stress magnitudes experienced by endothelial cells remain unknown. Motivated by the hypothesis that endothelial tip cells can experience physiological shear stress, the objective of this study was to estimate shear stress magnitudes due to local interstitial flow along endothelial tip cells that extend past capillary sprout lumens. A computational fluid dynamics model was used to estimate flows within a blind-ended vessel, transendothelial flow across the vessel wall, and flow within the surrounding perivascular space. The two-dimensional axisymmetric model consisted of four regions: vessel lumen, vessel wall, perivascular space, and interstitium. The vessel wall was assumed to be porous media with 0.3 μm thickness. Blood plasma was represented as an incompressible Newtonian fluid with density and viscosity equal to 1050 kg/m3 and 1.2 cP, respectfully. Shear stresses along the wall of the tip cell extending past the lumen were calculated for varying sprout lengths (50, 200, and 400 μm), perivascular space channel width (1, 4, and 9 μm), and hydraulic conductivities (1.35 and 6.75 x10-10 ms-1 Pa-1). Model parameters and geometric dimensions were selected based on previously reported literature values. Velocity profiles confirmed transmural flow across the vessel wall, nearly parabolic flow within the perivascular space, and velocity gradients along the tip cell. Increasing sprout length, increasing permeability, and decreasing perivascular space increased shear stress values with the perivascular space parameter being the major influencer. Maximum shear stresses along tip cells were consistently higher than the stresses within the sprout lumen. Wall shear stresses within the lumen ranged from 0.02 to 0.52 dyne/cm2 at the sprout entrance and linearly decreased to near zero along the sprout length. Tip cell wall shear stress due to interstitial flow ranged from 0.19 to 4.65 dyne/cm2. The results suggest that wall shear stress values along tip cells can reach physiological relevant values depending on parameter combinations. Our computational estimates motivate new questions related to whether increased shear stresses along tip cells versus luminal cells contribute phenotypic and function signatures of tip cells.

Neurotensin: A novel mediator of ovulation?

The midcycle luteinizing hormone (LH) surge initiates a cascade of events within the ovarian follicle which culminates in ovulation. Only mural granulosa cells and theca cells express large numbers of LH receptors, and LH-stimulated paracrine mediators communicate the ovulatory signal within the follicle. Recent reports identified the neuropeptide neurotensin (NTS) as a product of granulosa cells. Here, we demonstrate that granulosa cells were the primary site of NTS expression in macaque ovulatory follicles. Granulosa cell NTS mRNA and protein increased after human chorionic gonadotropin (hCG) administration, which substitutes for the LH surge. To identify ovulatory actions of NTS, a NTS-neutralizing antibody was injected into preovulatory macaque follicles. hCG administration immediately followed, and ovaries were removed 48hours later to evaluate ovulatory events. Follicles injected with control IgG ovulated normally. In contrast, 75% of NTS antibody-injected follicles failed to ovulate, containing oocytes trapped within unruptured, hemorrhagic follicles. Serum progesterone was unchanged. Of the three NTS receptors, SORT1 was highly expressed in follicular granulosa, theca, and endothelial cells; NTSR1 and NTSR2 were expressed at lower levels. Excessive blood cells in NTS antibody-injected follicles indicated vascular anomalies, so the response of monkey ovarian endothelial cells to NTS was evaluated in vitro. NTS stimulated endothelial cell migration and capillary sprout formation, consistent with a role for NTS in vascular remodeling associated with ovulation. In summary, we identified NTS as a possible paracrine mediator of ovulation. Further investigation of the NTS synthesis/response pathway may lead to improved treatments for infertility and novel targets for contraception.

Abstract A14: Modeling drug combination sensitivity with Hopfield networks and transcriptomics data

Abstract The Hopfield neural network model is one of the simplest models able to mathematically implement Waddington’s interpretation of normal and anomalous cell phenotypes as dynamical attractors of epigenetic landscapes. Here, we propose a computational approach based on Hopfield’s associative memories that integrate gene expression data and gene interactome networks in (1) a model representing the dynamics and control of disease progression in multiple myeloma (MM), and (2) a model describing the control of angiogenesis. The MM model is built using single-cell RNA-seq data from bone marrow aspirates of MM patients as well as patients diagnosed with monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM), two medical conditions that often progress to full MM. We identify different clusters of MGUS, SMM, and MM cells, map them to Hopfield associative memory patterns, and model the dynamics of transition between the different patterns. The model is then used to identify combinations of genes whose simultaneous inhibition is associated with delayed disease progression. In the angiogenesis control model, we use single-cell RNA-seq data to predict specific combinations of targets for inhibition that could induce a cellular transition from tip-like to stalk-like endothelial cells, inhibiting the formation of capillary sprouts in blood vessels. The model generates novel hypotheses for combinations that could complement standard VEGF inhibitors and lead to a more efficient control of angiogenesis in cancer. Citation Format: Carlo Piermarocchi, Sergii Domanskyi, Alex Hakansson, Giovanni Paternostro. Modeling drug combination sensitivity with Hopfield networks and transcriptomics data [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr A14.

Estimation of Shear Stress Values Along Endothelial Tip Cells Past the Lumen of Capillary Sprouts

Shear stress is commonly recognized as a regulator of endothelial cell dynamics associated with angiogenesis. A full understanding of how shear stresses influence endothelial cell phenotype and function during angiogenesis requires the identification of local stress distribution along capillary sprouts. However, the actual shear stress magnitudes experienced by endothelial cells remain unknown in large part to the difficulty in experimentally measuring local velocity profiles. The objective of this study was to estimate shear stress magnitudes due to local interstitial flow along endothelial tip cells that extend past capillary sprout lumens. A computational fluid dynamics model was used to estimate flows within a blind‐ended vessel, transendothelial flow out of through the vessel wall, and flow through a perivascular space. The two dimensional axisymmetric model consisted of 4 regions: vessel lumen, vessel wall, perivascular space and interstitium. The vessel wall was assumed to be porous media with 1 μm thickness. Blood plasma was represented as an incompressible Newtonian fluid with density and viscosity equal to 1050 kg/m3 and 1.2 cp, respectfully. Model parameters and geometric dimensions were selected based on previously reported literature values. Shear stresses along the wall of the tip cell extended past the lumen were calculated for varying sprout lengths (100, 200, and 500 μm), perivascular space channel width (1, 4, and 9 μm) and sprout permeability (1×10−18 and 1×10−17 m2). For the 200 μm sprout length scenario, shear stress values ranged from 0.009 to 5 dyne/cm2. Increasing sprout length, increasing permeability and decreasing perivascular space served to increase shear stress values. These results suggest the potential for endothelial cells past the lumen of a capillary sprout to experience negligible to stimulatory shear stresses dependent on sprout wall permeability and interstitial flow pathways.

Effects of Anti‐Cancer Therapy on Human Microvascular Function ‐ a Longitudinal Study

RationalCardiovascular toxicities that result from anti‐cancer therapy (CT) significantly contribute to the morbidity and mortality of breast cancer survivors. While the negative impact of CTs such as trastuzumab or doxorubicin on cardiomyocytes is well‐established, significantly less is known about how the circulatory system is affected. Impaired microvascular (MV) function as measured by coronary flow reserve is a recognized predictor of future adverse cardiovascular events. Our goal is to understand how CT damages the microcirculation and how that damage contributes to acute and long‐term cardiovascular toxicities.MethodsTo study the direct effect of CT on the MV function, we are conducting a longitudinal study of neoadjuvant breast cancer patients. Patient adipose samples are collected before, during, and after CT treatment. Endothelial‐dependent dilation to flow (FMD) and acetylcholine (Ach) and smooth muscle‐dependent dilation to papaverine were evaluated in freshly isolated microvessels via video microscopy. NO and mitochondrial ROS were evaluated via florescence microscopy. Data are presented as a percentage of maximum diameter and are analyzed with ANOVA RM with statistical significance set at *p<0.05; data are expressed as mean ± standard error. Additionally, angiogenic potential was evaluated using a matrigel plug assay. Data are presented as a percentage of wells with capillary sprouts. Cardiac function was monitored via standard clinical echocardiogram.ResultsVessels from breast cancer patients showed normal FMD and Ach responses and a normal increase in NO production to flow stimulus before CT; however, CT resulted in impairment of FMD (pre‐CT 80.9±4.2%; during‐CT 21.8%±3.1%*; post‐CT 30.3±4.5%*) and dilation to Ach (pre‐CT 81.3±9.1%; during‐CT 23.1%±1.8%*; post‐CT 38.6±6.5%*). Smooth muscle‐dependent dilation (to papaverine) was not affected by CT treatments. Angiogenic potential was impaired in adipose samples collected during CT (6.5%±6.5%) compared to pre‐CT (47.3%±10.4%). Surprisingly, despite significantly decreased endothelial function, angiogenic potential completely recovered one month after final CT treatment (59.8%±16.4%). No clinical signs of reduced cardiac function were observed in any of our study participants.ConclusionOur study shows for the first time a longitudinal evaluation of human microvascular function in response to anti‐cancer therapy. We observed significant reductions in MV function during treatment with CT in the absence of a clinically relevant reduction in cardiac function. Our findings suggest that CT‐induced MV dysfunction precedes and may contribute to future adverse CV events.Support or Funding InformationThis work was supported by NIH R01 HL133029, We Care Foundation Grant; MCWCardiovascular Center Pre‐PPG grant, Advancing a Healthier Wisconsin – Redox Biology Grant.

VE-Cadherin and Anastomosis of Blood Vessels Formed by Dental Stem Cells

It is known that dental pulp stem cells (DPSCs) can be induced to differentiate into vasculogenic endothelial (VE) cells. However, the process that results in sprouting and anastomosis of DPSC-derived vessels remains unclear. Here, we performed studies to understand the mechanisms underpinning the anastomosis of the host vasculature with blood vessels generated by DPSCs (a model for mesenchymal stem cells). VE-cadherin–silenced primary human DPSCs seeded in tooth slice/scaffolds and transplanted into the subcutaneous space of immunodeficient mice generated fewer functional blood vessels (i.e., anastomosed with the host vasculature) than control DPSCs transduced with scrambled sequences. Both VE-cadherin–silenced and mitogen-activated protein kinase kinase 1 (MEK1)–silenced cells showed a decrease in the number of capillary sprouts in vitro. Interestingly, DPSC stably transduced with a VE-cadherin reporter demonstrated that vascular endothelial growth factor (VEGF) induces VE-cadherin expression in sprouting DPSCs undergoing anastomosis, but not in quiescent DPSCs. To begin to understand the mechanisms regulating VE-cadherin, we stably silenced MEK1 and observed that VEGF was no longer able to induce VE-cadherin expression and capillary sprout formation. Notably ERG, a transcriptional factor downstream from MEK/ERK, binds to the promoter region of VE-cadherin (chip assay) and is induced by VEGF in DPSCs. Collectively, these data defined a signaling pathway triggered by VEGF that results in phosphorylation of MEK1/ERK and activation of ERG leading to expression of VE-cadherin, which is required for anastomosis of DPSC-derived blood vessels. In conclusion, these results unveiled a signaling pathway that enables the generation of functional blood vessels upon vasculogenic differentiation of DPSCs.

Thrombospondin 1 (THBS1) Promotes Follicular Angiogenesis, Luteinization, and Ovulation in Primates.

Angiogenesis is essential to both ovulation and the formation of the corpus luteum. The thrombospondin (THBS) family of glycoproteins plays diverse roles in regulation of angiogenesis, but the role of these vascular regulators in ovulation and luteinization remain to be elucidated. Using the cynomolgus macaque as a model for human ovulation, we demonstrated that levels of THBS1 mRNA and protein in preovulatory follicle granulosa cells increased after the ovulatory gonadotropin surge, with peak levels just before the expected time of ovulation. THBS1 treatment of monkey ovarian microvascular endothelial cells in vitro stimulated migration, proliferation, and capillary sprout formation, consistent with a pro-angiogenic action of THBS1. Injection of an anti-THBS1 antibody into monkey preovulatory follicles reduced rates of follicle rupture and oocyte release in response to an ovulatory gonadotropin stimulus when compared with control IgG-injected follicles. Interestingly, two of three oocytes from anti-THBS1 antibody injected follicles were germinal vesicle intact, indicating that meiosis failed to resume as anticipated. Follicles injected with anti-THBS1 antibody also showed reduced granulosa cell layer expansion, endothelial cell invasion, and capillary formation when compared to control IgG-injected follicles. Overall, these findings support a critical role for THBS1 in follicular angiogenesis, with implications for both successful ovulation and corpus luteum formation.

Analysis of a chemotaxis-convection model of capillary-sprout growth during tumor angiogenesis

In a bounded real interval, this work considers the chemotaxis-convection model{ut=d1uxx−(uvx)x+(uwx)x,vt=d2vxx+(vwx)x+ku−μv,wt=d3wxx+ru−δw, that describes the branching of capillary sprouts during angiogenesis, where d1,d2,d3,k,r,μ and δ are positive parameters. It is shown that for all suitably regular initial data, an associated Neumann initial-boundary problem admits a globally defined bounded smooth solution.

EphrinB2/EphB4 Signaling Regulates DPSCs to Induce Sprouting Angiogenesis of Endothelial Cells

Dental pulp stem cells (DPSCs) are capable of facilitating angiogenesis resembling pericytes when located adjacent to endothelial cells (ECs). Nevertheless, the precise mechanisms orchestrating their proangiogenic functions remain unclear. Using a 3-dimensional (3-D) fibrin gel model, we aimed to investigate whether EphrinB2/EphB4 signaling in DPSCs plays a role in supporting vascular morphogenesis mediated by ECs, together with the underlying mechanism involved. The EphrinB2/EphB4 signaling was inhibited either by a pharmacological inhibitor of EphB4 receptor or by knocking down the expressions of EphrinB2 and EphB4 using lentiviral small hairpin RNA (shRNA). DPSCs were either encapsulated in fibrin gel together with human umbilical vein endothelial cells (HUVECs) or cultured as a monolayer on top of HUVECs to investigate both paracrine and juxtacrine interactions simultaneously. Following 10 d of direct coculture, we found that pharmacological inhibition of EphrinB2/EphB4 signaling severely impaired vessel formation and laminin deposition. When directly cocultured with HUVECs, knockdown of EphrinB2 or EphB4 in DPSCs significantly inhibited endothelial sprouting, resulting in less capillary sprouts with reduced vessel length (P < 0.05). By contrast, when DPSCs were not in direct contact with HUVECs, attenuation of EphrinB2 or EphB4 expression levels in DPSCs did not exert any significant effects on capillary morphogenesis. Noticeably, exogenous stimulation with soluble EphrinB2-Fc or EphB4-Fc (1 µg/mL) enhanced vascular endothelial growth factor (VEGF) secretion from DPSCs, thereby moderately promoting angiogenic cascades in the fibrin matrix. This study, for the first time, reveals a crucial role of EphrinB2/EphB4 signaling in regulating the capacity of DPSCs to induce sprouting angiogenesis. These findings advance our understanding of postnatal angiogenesis and may have future regenerative medicine applications.