Mature Pericytes Research Articles

The development of brain pericytes requires expression of the transcription factor nkx3.1 in intermediate precursors.

Brain pericytes are one of the critical cell types that regulate endothelial barrier function and activity, thus ensuring adequate blood flow to the brain. The genetic pathways guiding undifferentiated cells into mature pericytes are not well understood. We show here that pericyte precursor populations from both neural crest and head mesoderm of zebrafish express the transcription factor nkx3.1 develop into brain pericytes. We identify the gene signature of these precursors and show that an nkx3.1-, foxf2a-, and cxcl12b-expressing pericyte precursor population is present around the basilar artery prior to artery formation and pericyte recruitment. The precursors later spread throughout the brain and differentiate to express canonical pericyte markers. Cxcl12b-Cxcr4 signaling is required for pericyte attachment and differentiation. Further, both nkx3.1 and cxcl12b are necessary and sufficient in regulating pericyte number as loss inhibits and gain increases pericyte number. Through genetic experiments, we have defined a precursor population for brain pericytes and identified genes critical for their differentiation.

Abstract LB053: PDAC derived exosomes manipulate tumor pericyte phenotype

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in men and women in the U.S., with a 5-year survival rate of 10%. This poor prognosis is partly due to the aggressive nature of the disease, lack of early detection methods, its complex and dense tumor microenvironment (TME), and lack of effective treatment options. Within the PDAC TME, morphologically aberrant leaky vessels are responsible for hypoxia and impaired immune response, which likely reduces the efficacy of cancer therapies. We and others showed that pericyte coverage significantly correlates with vascular integrity/function and intratumoral hypoxia. Therefore, understanding perivascular heterogeneity and the unique contribution of each phenotype to the TME evolution is a critical step towards personalized treatment options for PDAC. Our study revealed that tumor-associated pericytes across all PDAC tumor tissues exhibited ectopic αSMA expression up to 10X higher compared to the normal pericytes. This aberrant pericyte phenotype was correlated with vascular leakiness and hypoxia, whereas the Des+ mature pericyte phenotype was inversely correlated with vessel leakiness. Our attempt to elucidate the underlying mechanism of pericyte phenotype switching using in vitro culture system shows that pancreatic cancer cell-derived extracellular vesicles (PC-Exo) is a potent inducer of αSMA expression in pericyte. PC-Exo was also sufficient enough to induce αSMA expression in normal pericytes within the pancreas when injected into the WT mice. In addition to the changes in pericyte markers, PC-Exo stimulated pericytes exhibited immunomodulatory phenotype with significantly higher expression of HLA-DRA, CD274, CD80, E-selectin, and P-selectin. Considering αSMA is a cytoskeletal protein that can influence cellular stiffness, migration, and downstream signaling, we also examined the biomechanical properties of pericytes with altered phenotype. The results show a significant increase in pericyte stiffness and cellular height of aSMA+ pericyte. These pericytes also have elongated morphology and cytoskeletal reorganization visualized by αSMA and phalloidin immunostaining. A recent study showed that acquired αSMA expression converts capillary pericytes into contractile vascular smooth muscle-like cells. Blood vessels are constricted and collapsed within the PDAC TME, which may be partly caused by an abundance of αSMA+ pericytes. In conclusion, our study indicates that tumor-associated pericytes undergo phenotype switching under the influence of pancreatic cancer cells, and these aberrant pericytes might contribute to non-optimal vascular integrity/function. Together, our study indicates that tumor conditioned αSMA+ pericytes present mechanical abnormalities and immune-suppressive features. Future work includes exploring the vascular normalization approach by suppressing pericyte phenotype switching to enhance vascular function and chemo- and immunotherapeutic efficacy. Citation Format: Vikneshwari Natarajan, Alexander Delgado, Reed Jacobson, Lina Alhalhooly, Yongki Choi, Sangdeuk Ha, Jiha Kim. PDAC derived exosomes manipulate tumor pericyte phenotype [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 LB053.

CD146+ Pericytes Subset Isolated from Human Micro-Fragmented Fat Tissue Display a Strong Interaction with Endothelial Cells: A Potential Cell Target for Therapeutic Angiogenesis.

Pericytes (PCs) are mesenchymal stromal cells (MSCs) that function as support cells and play a role in tissue regeneration and, in particular, vascular homeostasis. PCs promote endothelial cells (ECs) survival which is critical for vessel stabilization, maturation, and remodeling. In this study, PCs were isolated from human micro-fragmented adipose tissue (MFAT) obtained from fat lipoaspirate and were characterized as NG2+/PDGFRβ+/CD105+ cells. Here, we tested the fat-derived PCs for the dispensability of the CD146 marker with the aim of better understanding the role of these PC subpopulations on angiogenesis. Cells from both CD146-positive (CD146+) and negative (CD146−) populations were observed to interact with human umbilical vein ECs (HUVECs). In addition, fat-derived PCs were able to induce angiogenesis of ECs in spheroids assay; and conditioned medium (CM) from both PCs and fat tissue itself led to the proliferation of ECs, thereby marking their role in angiogenesis stimulation. However, we found that CD146+ cells were more responsive to PDGF-BB-stimulated migration, adhesion, and angiogenic interaction with ECs, possibly owing to their higher expression of NCAM/CD56 than the corresponding CD146− subpopulation. We conclude that in fat tissue, CD146-expressing cells may represent a more mature pericyte subpopulation that may have higher efficacy in controlling and stimulating vascular regeneration and stabilization than their CD146-negative counterpart.

Transcriptomic Profiling of Dental Pulp Pericytes: An RNAseq Approach

Pericytes represent a population of mesenchymal cells that are found in virtually all vascularised organs where they share a range of similar features, mostly associated with vascular maintenance roles. It is becoming increasingly apparent that organ to organ differences exist between pericytes that directly relate to tissue-specific functions of these cells. Here, we present novel data indicating that in dental structures, pericytes have an immunomodulatory role. We show evidence supporting the concept that pericytes are a source of multiple cytokines and chemokines within the dental pulp, capable of regulating the extravasation of circulating lymphocytes. Using single cell RNA-seq we show that this is attributed to a defined pericyte sub-population that exists in vivo. In addition, as a consequence of our transcriptomic screen, we detect and validate a novel marker that can identify mature pericytes that can give rise to odontoblasts in vivo. Using transcriptomic screening, this marker is also shown to be expressed in pericytes residing in multiple other tissues including bone marrow, lung, and pancreas.

Abstract 5097: Pancreatic cancer cells derived exosomes stimulate pericyte phenotype switching in the tumor microenvironment

Abstract In pancreatic cancer (PC), most conventional and targeted therapies fail to provide substantial response partly due to the limited delivery efficacy of cytotoxic drugs. Poor vascular structure and stroma-rich tumor microenvironment (TME) likely contribute to such failure. We have previously shown that in breast cancer, differential perivascular investment significantly impacts vascular functionality and drug delivery efficacy. Considering unique pancreatic cancer TME, it is a promising approach to "re-invest" perivascular cells in which blood vessels are in their optimal functional status. As a first attempt to understand the perivascular phenotype of pancreatic tumors, we utilized a multiplex imaging analysis technique to map out differential pericyte markers expression on several mouse model systems for PC as well as tumor microarray (TMA) of patients with PC. Among pericyte markers examined, alpha-smooth muscle actin (α-SMA) expression was significantly increased in all cases of PC compared to normal pancreas tissues, which skewed the ratio between Desmin (mature pericyte marker) and α-SMA. To determine if eliminating pathological pericytes can normalize vascular phenotype of pancreatic tumor, we utilized PDGFRβ-TK transgenic mice in which proliferating pericytes that express PDGFRβ are depleted. However, eliminating these pericytes resulted in increased metastasis accompanied by increased vascular leakiness. This result indicates that elimination tumor-associated pericyte might result in an adverse effect instead of vascular normalization. Therefore, we hypothesize that normalizing vascular phenotype by re-investment of perivascular cells will likely contribute to improving TME for a better outcome. To understand the molecular mechanism governing pericyte phenotype switching, we established in vitro vascular cell culture system. The results indicate that PC cells derived exosomes can modify pericyte phenotype that resembles tumor-associated pericyte. Such phenotype conversion was even further enhanced in a hypoxic environment. Currently, we are investigating global gene expression changes of pericyte upon exposure to PC cell exosomes using a single-cell RNA sequencing method to have a comprehensive understanding of pericyte phenotype switching. Currently, we are performing in vivo perivascular re-investment using our candidate molecules to prove our hypothesis that perivascular re-investment will improve vascular performance and consequently enhance drug delivery efficacy. Citation Format: Alexander Delgado, Reed Jacobson, Dylan Wrede, Sangdeuk Ha, Jiha Kim. Pancreatic cancer cells derived exosomes stimulate pericyte phenotype switching in the tumor microenvironment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5097.

Phosphoinositide 3-Kinase-Regulated Pericyte Maturation Governs Vascular Remodeling.

Pericytes regulate vessel stabilization and function, and their loss is associated with diseases such as diabetic retinopathy or cancer. Despite their physiological importance, pericyte function and molecular regulation during angiogenesis remain poorly understood. To decipher the transcriptomic programs of pericytes during angiogenesis, we crossed Pdgfrb(BAC)-CreERT2 mice into RiboTagflox/flox mice. Pericyte morphological changes were assessed in mural cell-specific R26-mTmG reporter mice, in which low doses of tamoxifen allowed labeling of single-cell pericytes at high resolution. To study the role of phosphoinositide 3-kinase (PI3K) signaling in pericyte biology during angiogenesis, we used genetic mouse models that allow selective inactivation of PI3Kα and PI3Kβ isoforms and their negative regulator phosphate and tensin homolog deleted on chromosome 10 (PTEN) in mural cells. At the onset of angiogenesis, pericytes exhibit molecular traits of cell proliferation and activated PI3K signaling, whereas during vascular remodeling, pericytes upregulate genes involved in mature pericyte cell function, together with a remarkable decrease in PI3K signaling. Immature pericytes showed stellate shape and high proliferation, and mature pericytes were quiescent and elongated. Unexpectedly, we demonstrate that PI3Kβ, but not PI3Kα, regulates pericyte proliferation and maturation during vessel formation. Genetic PI3Kβ inactivation in pericytes triggered early pericyte maturation. Conversely, unleashing PI3K signaling by means of PTEN deletion delayed pericyte maturation. Pericyte maturation was necessary to undergo vessel remodeling during angiogenesis. Our results identify new molecular and morphological traits associated with pericyte maturation and uncover PI3Kβ activity as a checkpoint to ensure appropriate vessel formation. In turn, our results may open new therapeutic opportunities to regulate angiogenesis in pathological processes through the manipulation of pericyte PI3Kβ activity.

Autophagy modulation altered differentiation capacity of CD146+ cells toward endothelial cells, pericytes, and cardiomyocytes

BackgroundTo date, many attempts are employed to increase the regenerative potential of stem cells. In this study, we evaluated the hypothesis of whether an autophagy modulation could alter differentiation potency of CD146+ cells into mature pericyte, endothelial, and cardiomyocyte lineage.MethodsIn this study, CD146+cells were enriched from the human bone marrow aspirates and trans-differentiated into mature endothelial cells, pericytes, and cardiomyocytes after exposure to autophagy stimulator (50-μM Met)/inhibitor (15-μM HCQ). The protein levels of autophagy proteins were monitored by western blotting. NO content was measured using the Griess assay. Using real-time PCR assay and western blotting, we monitored the lineage protein and gene levels. Pro-inflammatory cytokine and angiocrine factors were measured by ELISA. The fatty acid change was determined by gas chromatography. We also measured exosome secretion capacity by measuring AChE activity and real-time PCR assay.ResultData revealed the modulation of autophagy factors, Beclin-1, P62, and LC3 II/I ratio in differentiating CD146+ cells after exposure to Met and HCQ (p < 0.05). The inhibition of autophagy increased NO content compared to the Met-treated cells (p < 0.05). Real-time PCR analysis showed that the treatment of CD146+ cells with autophagy modulators altered the expression of VE-cadherin, cTnI, and α-SMA (p < 0.05). Met increased the expression of VE-cadherin, α-SMA, and cTnI compared to the HCQ-treated cells (p < 0.05) while western blotting revealed the protein synthesis of all lineage-specific proteins under the stimulation and inhibition of autophagy. None statistically significant differences were found in the levels of Tie-1, Tie-2, VEGFR-1, and VEGFR-2 after autophagy modulation. Fatty acid profile analysis revealed the increase of unsaturated fatty acids after exposure to HCQ (p < 0.05). The treatment of cells with HCQ increased the levels of TNF-α and IL-6 compared to the Met-treated cells. Data revealed the increase of exosome biogenesis and secretion to the supernatant in cells treated with HCQ compared to the Met groups (p < 0.05).ConclusionsIn summary, autophagy modulation could alter differentiation potency of CD146+cells which is important in cardiac regeneration.

Pancreatic pericytes originate from the embryonic pancreatic mesenchyme

The embryonic origin of pericytes is heterogeneous, both between and within organs. While pericytes of coelomic organs were proposed to differentiate from the mesothelium, a single-layer squamous epithelium, the embryonic origin of pancreatic pericytes has yet to be reported. Here, we show that adult pancreatic pericytes originate from the embryonic pancreatic mesenchyme. Our analysis indicates that pericytes of the adult mouse pancreas originate from cells expressing the transcription factor Nkx3.2. In the embryonic pancreas, Nkx3.2-expressing cells constitute the multilayered mesenchyme, which surrounds the pancreatic epithelium and supports multiple events in its development. Thus, we traced the fate of the pancreatic mesenchyme. Our analysis reveals that pancreatic mesenchymal cells acquire various pericyte characteristics, including gene expression, typical morphology, and periendothelial location, during embryogenesis. Importantly, we show that the vast majority of pancreatic mesenchymal cells differentiate into pericytes already at embryonic day 13.5 and progressively acquires a more mature pericyte phenotype during later stages of pancreas organogenesis. Thus, our study indicates the embryonic pancreatic mesenchyme as the primary origin to adult pancreatic pericytes. As pericytes of other coelomic organs were suggested to differentiate from the mesothelium, our findings point to a distinct origin of these cells in the pancreas. Thus, our study proposes a complex ontogeny of pericytes of coelomic organs.

Abstract 6: Role of Notch signaling in bevacizumab-induced vascular normalization in glioblastoma

Abstract Tumor angiogenesis occurs in the setting of a defective vasculature, which is associated with increased vascular permeability and enhanced tumor permeability. Bevacizumab is used to treat malignant glioma and was found to reduce microvascular density and prune abnormal tumor microvessels. Using electron microscopic observation of two autopsy cases, we investigated the effects of blood vessel normalization in glioblastomas treated with bevacizumab. Notch-1 and SMA immunostaining were used to compare initial surgical specimens with postmortem specimens obtained after bevacizumab treatment. Postmortem samples showed marked proliferation of SMA-positive cells (pericytes) in tumor vessels and marked proliferation of Notch-1–positive cells around vessels. Electron microscopic images confirmed the presence of pericytes surrounding the vascular endothelium. These findings suggest that bevacizumab treatment promotes vascular normalization by recruiting mature pericytes. Next, we investigated the effects of bevacizumab on VEGF inhibition in glioma stem cells. Bevacizumab treatment attenuated activation of VEGFR2 and increased Notch signaling expression. VEGF inhibition by bevacizumab treatment attenuated proliferation and self-renewal of glioma stem cells and induced endothelial and pericyte differentiation. In tumor angiogenesis, vascular endothelial growth factor induces sprouting angiogenesis and recruitment of vascular endothelial cells such as tip cells, stalk cells, and phalanx cells. Fully mature phalanx cells are in close contact with pericytes. Our results suggest that bevacizumab treatment induces glioma stem cells to differentiate to endothelium and pericytes. These mechanisms might be important in normalizing tumor vasculature after bevacizumab treatment and could be useful in improving the effectiveness of current glioma therapy. Citation Format: Norihiko Saito, Kazuya Aoki, Nozomi Hirai, Satoshi Fujita, Haruo Nakayama, Morito Hayashi, Takatoshi Sakurai, Satoshi Iwabuchi. Role of Notch signaling in bevacizumab-induced vascular normalization in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 6.

Aβ inhibits mesenchymal stem cell-pericyte transition through MAPK pathway.

Multiple independent reports have demonstrated pericyte loss in both the hippocampus and cortex in human Alzheimer's disease (AD). The differentiation and recruitment of pericytes are the essential steps in vasculature development. However, the role of amyloid beta (Aβ) in pericyte differentiation has not yet been fully elucidated. In this study, we investigated the interaction between Aβ and differentiation of mesenchymal stem cells (MSCs) toward pericytes in culture. Our results showed that mice overexpressing Aβ-precursor protein (APP/PS1) exhibited the loss of pericytes compared with the control group mice, evidenced by the lack of desmin expression in the cortex of 12-month-old mice. Interestingly, we further found that both Aβ40 and Aβ42 inhibited the expressions of pericyte markers (α-SMA, desmin, and PDGFRβ) in cultured MSCs which can be differentiated into mature pericytes. Mechanistically, the inhibitory effects of Aβs on MSC-pericyte transition is mediated by the activation of the ERK1/2 MAPK signal pathway. These new insights into the roles of Aβ in pericyte differentiation may help to develop more effective strategies for the treatment of AD.

New Pericytes in the Adult Brain Originate from a Previously Unrecognized Pericyte Precursor

Pericytes are essential for maintaining the stability of CNS blood-vessels and the blood-brain- barrier. They are closely associated with endothelia and can be identified by a set of markers. New pericytes are required during neo-angiogenesis but how pericytes are generated in the adult brain is unknown. Here, we investigated neoplastic vascularization of the adult brain in a transgenic lineage-tracing model. We observed that mature pericytes largely originate from a newly identified precursor celltype, which is distinct from established brain cell-populations as evidenced by single-cell RNAseq studies. These precursors are characterized as pericyte marker-negative cells of non-hematopoietic origin, which are distant from blood-vessels and highly proliferative. Pericyte precursors emerge also in developmental angiogenesis and lineage ablation experiments in brain tumor models showed that they are necessary to generate new, mature pericytes supporting tumor expansion. Overall, pericyte precursors control vascular plasticity and provide an excellent therapeutic target for brain tumors.

Abstract 774: Notch pathway activation predicts resistance to bevacizumab therapy in glioblastoma

Abstract Glioblastoma, the most common adult glioma, is associated with a dismal prognosis. Treatment with bevacizumab has not significantly prolonged overall patient survival times. Glioblastoma resistance to angiogenesis inhibitors is attributed to multiple interacting mechanisms. We have thus embarked on a comprehensive effort to detecting expression signatures that are associated with response to the therapy and these signatures may allow prospective selection of patients with high likelihood of responding to therapy. Notch signaling pathway is an evolutionarily conserved pathway that plays an important role in multiple cellular and developmental processes including cell fate decision, differentiation, proliferation, survival, angiogenesis and migration. Analysis of The Cancer Genome Atlas expression dataset identified a group (43.9%) of tumors with proneural signature showing high Notch pathway activation. In this study, we compared CD133, Notch, and VEGF expressions in histological sections of primary and recurrent glioblastomas after radiotherapy and chemotherapy. Tumor samples were collected from 27 patients at the time of tumor recurrence. We used immunohistochemical techniques to compare expression of CD133, Notch-1 and VEGF. Expressions of CD133-, Notch-1-, and VEGF-positive glioma cells were higher in recurrent glioblastoma after radiotherapy and chemotherapy. To determine the clinical importance of Notch-1 expression in glioblastoma, we analyzed 15 patients who had received bevacizumab therapy followed by a second surgery at recurrence. OS was significantly longer in cases with Notch-1 negativity (8.8 months) than in those with Notch-1 positivity (6.8 months). Electron microscopic observation of two autopsy cases revealed the effects of blood vessel normalization in Notch-1 positive glioblastoma. Electron microscopic images confirmed the presence of pericytes surrounding the vascular endothelium. Autopsied tumors exhibited marked proliferation of Notch-1 and VEGF positive cells around vessels. In tumor angiogenesis, vascular endothelial growth factor and Notch signaling induce sprouting angiogenesis and recruitment of vascular endothelial cells such as tip cells, stalk cells, and phalanx cells. Fully mature phalanx cells are in close contact with pericytes. These findings indicate that bevacizumab treatment promotes vascular normalization by recruiting mature pericytes and associated with resistance to bevacizumab therapy in glioblastoma with high Notch pathway activation. Citation Format: Norihiko Saito, Kazuya Aoki, Nozomi Hirai, Satoshi Fujita, Junya Iwama, Masashi Ikota, Haruo Nakayama, Morito Hayashi, Keisuke Ito, Takatoshi Sakurai, Satoshi Iwabuchi. Notch pathway activation predicts resistance to bevacizumab therapy in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 774. doi:10.1158/1538-7445.AM2017-774

Astrocytes at the Hub of the Stress Response: Potential Modulation of Neurogenesis by miRNAs in Astrocyte-Derived Exosomes.

Repetitive stress negatively affects several brain functions and neuronal networks. Moreover, adult neurogenesis is consistently impaired in chronic stress models and in associated human diseases such as unipolar depression and bipolar disorder, while it is restored by effective antidepressant treatments. The adult neurogenic niche contains neural progenitor cells in addition to amplifying progenitors, neuroblasts, immature and mature neurons, pericytes, astrocytes, and microglial cells. Because of their particular and crucial position, with their end feet enwrapping endothelial cells and their close communication with the cells of the niche, astrocytes might constitute a nodal point to bridge or transduce systemic stress signals from peripheral blood, such as glucocorticoids, to the cells involved in the neurogenic process. It has been proposed that communication between astrocytes and niche cells depends on direct cell-cell contacts and soluble mediators. In addition, new evidence suggests that this communication might be mediated by extracellular vesicles such as exosomes, and in particular, by their miRNA cargo. Here, we address some of the latest findings regarding the impact of stress in the biology of the neurogenic niche, and postulate how astrocytic exosomes (and miRNAs) may play a fundamental role in such phenomenon.

Optical coherence tomography angiography in age-related macular degeneration: persistence of vascular network in quiescent choroidal neovascularization.

Optical coherence tomography angiography in age-related macular degeneration: persistence of vascular network in quiescent choroidal neovascularization.

Negative pressure environment promotes angiogenesis and microvessel maturation

Objective To study the effect of negative pressure environment on angiogenesis and microvessel maturation and relevant signaling pathway, and explore the potential mechanisms of angiogenin/tyrosine kinase receptor-2 signal pathway promoting angiogenesis and microvessel maturation under negative pressure environment. Methods Successive 58 patients enrolled in this study were treated with negative pressure or petroleum gauze, and at 1st, 3rd, 7th, and 15th day blood flow perfusion was detected, as well as the Western boltting was applied to detect angiogenin-1 (Ang-1), the phosphorylation of tyrosine kinase receptor-2 (pTie-2), angiogenin-2 (Ang-2), α-smooth muscle actin (α-SMA) and collagen type Ⅳ respectively. Results Negative pressure environment could change the expression of related factorsin the early stages of wound healing (1-3 d) and late stage (7-15 d). In experimental group on day 3, the relative value of Ang-2 was 1.87±0.35 and significantly higher than control group (1.32±0.23, P<0.05), relative value of pTie-2 was 0.36±0.02 and significantly lower than control group (0.87±0.04, P<0.05), relative value of Ang-1 was 0.34±0.02 and significantly lower than control group (0.78±0.07, P<0.05), the ratio of Ang-1/Ang-2 was 0.34±0.02 and significantly lower than the control group (0.78±0.07, P<0.05). In the experimental group on day 15, negative pressure environment leadin to the relative value of Ang-1 was 1.89±0.24 and significantly higher than conrol group (1.35±0.22, P<0.05), relative value of α- smooth muscle actin was 2.56±0.42 and significantly higher than control group (1.68±0.31, P<0.05), relative value of collagen type Ⅳ was 2.32±0.35 and significantly higher than control group (1.36±0.24, P<0.05), the relative value of pTie-2 was 2.12±0.44 and significantly higher than control group (1.23±0.31, P<0.05), the ratio of Ang-1/Ang-2 was 4.86±0.67 and significantly higher than control group (1.87±0.54, P<0.05), the relative value of Ang-2 was 0.36±0.05 and significantly lower than control group (0.71±0.06, P<0.05). Conclusion Negative pressure could preferentially promote microvessel destabilization in the early stage of wound healing, consequently increase the number of angiogenesis. Subsequently, in the later stage of wound healing the negative pressure could preferentially promote microvessel stabilization, and thus promote microvessel maturation through the angiogenin/tyrosine kinase receptor-2 signal pathway. Negative pressure could increase blood flow perfusion because of abundant and mature microvessel pericytes in the wound. Key words: Negative pressure environment; Blood vessel maturation; Blood vessel stabilized; Blood flow perfusion

Lenalidomide normalizes tumor vessels in colorectal cancer improving chemotherapy activity.

BackgroundAngiogenesis inhibition is a promising approach for treating metastatic colorectal cancer (mCRC). Recent evidences support the seemingly counterintuitive ability of certain antiangiogenic drugs to promote normalization of residual tumor vessels with important clinical implications. Lenalidomide is an oral drug with immune-modulatory and anti-angiogenic activity against selected hematologic malignancies but as yet little is known regarding its effectiveness for solid tumors. The aim of this study was to determine whether lenalidomide can normalize colorectal cancer neo-vessels in vivo, thus reducing tumor hypoxia and improving the benefit of chemotherapy.MethodsWe set up a tumorgraft model with NOD/SCID mice implanted with a patient-derived colorectal cancer liver metastasis. The mice were treated with oral lenalidomide (50 mg/Kg/day for 28 days), intraperitoneal 5-fluorouracil (5FU) (20 mg/Kg twice weekly for 3 weeks), combination (combo) of lenalidomide and 5FU or irrelevant vehicle. We assessed tumor vessel density (CD146), pericyte coverage (NG2; alphaSMA), in vivo perfusion capability of residual vessels (lectin distribution essay), hypoxic areas (HP2-100 Hypoxyprobe) and antitumor activity in vivo and in vitro.ResultsTreatment with lenalidomide reduced tumor vessel density (p = 0.0001) and enhanced mature pericyte coverage of residual vessels (p = 0.002). Perfusion capability of tumor vessels was enhanced in mice treated with lenalidomide compared to controls (p = 0.004). Accordingly, lenalidomide reduced hypoxic tumor areas (p = 0.002) and enhanced the antitumor activity of 5FU in vivo. The combo treatment delayed tumor growth (p = 0.01) and significantly reduced the Ki67 index (p = 0.0002). Lenalidomide alone did not demonstrate antitumor activity compared to untreated controls in vivo or against 4 different mCRC cell lines in vitro.ConclusionsWe provide the first evidence of tumor vessel normalization and hypoxia reduction induced by lenalidomide in mCRC in vivo. This effect, seemingly counterintuitive for an antiangiogenic compound, translates into indirect antitumor activity thus enhancing the therapeutic index of chemotherapy. Our findings suggest that further research should be carried out on synergism between lenalidomide and conventional therapies for treating solid tumors that might benefit from tumor vasculature normalization.

Vascular phenotype identification and anti-angiogenic treatment recommendation: A pseudo-multiscale mathematical model of angiogenesis

The development of anti-angiogenic drugs for cancer therapy has yielded some promising candidates, but novel approaches for interventions to angiogenesis have led to disappointing results. In addition, there is a shortage of biomarkers that are predictive of response to anti-angiogenic treatments. Consequently, the complex biochemical and physiological basis for tumour angiogenesis remains incompletely understood. We have adopted a mathematical approach to address these issues, formulating a spatially averaged multiscale model that couples the dynamics of VEGF, Ang1, Ang2 and PDGF, with those of mature and immature endothelial cells and pericyte cells. The model reproduces qualitative experimental results regarding pericyte coverage of vessels after treatment by anti-Ang2, anti-VEGF and combination anti-VEGF/anti-Ang2 antibodies. We used the steady state behaviours of the model to characterise angiogenic and non-angiogenic vascular phenotypes, and used mechanistic perturbations representing hypothetical anti-angiogenic treatments to generate testable hypotheses regarding transitions to non-angiogenic phenotypes that depend on the pre-treatment vascular phenotype. Additionally, we predicted a synergistic effect between anti-VEGF and anti-Ang2 treatments when applied to an immature pre-treatment vascular phenotype, but not when applied to a normalised angiogenic pre-treatment phenotype. Based on these findings, we conclude that changes in vascular phenotype are predicted to be useful as an experimental biomarker of response to treatment. Further, our analysis illustrates the potential value of non-spatial mathematical models for generating tractable predictions regarding the action of anti-angiogenic therapies.

High-Dose, Single-Fraction Irradiation Rapidly Reduces Tumor Vasculature and Perfusion in a Xenograft Model of Neuroblastoma

To characterize the effects of high-dose radiation therapy (HDRT) on neuroblastoma tumor vasculature, including the endothelial cell (EC)-pericyte interaction as a potential target for combined treatment with antiangiogenic agents. The vascular effects of radiation therapy were examined in a xenograft model of high-risk neuroblastoma. In vivo 3-dimensional contrast-enhanced ultrasonography (3D-CEUS) imaging and immunohistochemistry (IHC) were performed. HDRT significantly reduced tumor blood volume 6 hours after irradiation compared with the lower doses used in conventionally fractionated radiation. There was a 63% decrease in tumor blood volume after 12-Gy radiation compared with a 24% decrease after 2 Gy. Analysis of tumor vasculature by lectin angiography showed a significant loss of small vessel ends at 6 hours. IHC revealed a significant loss of ECs at 6 and 72 hours after HDRT, with an accompanying loss of immature and mature pericytes at 72 hours. HDRT affects tumor vasculature in a manner not observed at lower doses. The main observation was an early reduction in tumor perfusion resulting from a reduction of small vessel ends with a corresponding loss of endothelial cells and pericytes.

A novel population of local pericyte precursor cells in tumor stroma that require Notch signaling for differentiation

Pericytes are perivascular support cells, the origin of which in tumor tissue is not clear. Recently, we identified a Tie1+ precursor cell that differentiates into vascular smooth muscle, in a Notch-dependent manner. To understand the involvement of Notch in the ontogeny of tumor pericytes we used a novel flow immunophenotyping strategy to define CD146+/CD45−/CD31−/lo pericytes in the tumor stroma. This strategy combined with ex vivo co-culture experiments identified a novel pericyte progenitor cell population defined as Sca1hi/CD146−/CD45−/CD31−. The differentiation of these progenitor cells was stimulated by co-culture with endothelial cells. Overexpression of the Notch ligand Jagged1 in endothelial cells further stimulated the differentiation of Sca1hi/CD146−/CD45−/CD31− cells into pericytes, while inhibition of Notch signaling with a γ-secretase inhibitor reduced this differentiation. However, Notch inhibition specifically in Tie1-expressing cells did not change the abundance of pericytes in tumors, suggesting that the pericyte precursor is distinct from the vascular smooth muscle cell precursor. Transplant experiments showed that the bone marrow contributes minimally to tumor pericytes. Immunophenotyping revealed that Sca1hi/CD146−/CD45−/CD31− cells have greater potential to differentiate into pericytes and have increased expression of classic mesenchymal stem cell markers (CD13, CD44, Nt5e and Thy-1) compared to Sca1−/lo/CD146−/CD45−/CD31− cells. Our results suggest that a local Sca1hi/CD146−/CD45−/CD31− pericyte progenitor resides in the tumor microenvironment and requires Notch signaling for differentiation into mature pericytes.

Nestin(+) tissue-resident multipotent stem cells contribute to tumor progression by differentiating into pericytes and smooth muscle cells resulting in blood vessel remodeling.

Tumor vessels with resistance to anti-angiogenic therapy are characterized by the normalization of the vascular structures through integration of mature pericytes and smooth muscle cells (SMC) into the vessel wall, a process termed vessel stabilization. Unfortunately, stabilization-associated vascular remodeling can result in reduced sensitivity to subsequent anti-angiogenic therapy. We show here that blockade of VEGF by bevacizumab induces stabilization of angiogenic tumor blood vessels in human tumor specimen by recruiting Nestin-positive cells, whereas mature vessels down-regulated Nestin-expression. Using xenograft tumors growing on bone-marrow (BM) chimera of C57Bl/6 wildtype and Nestin-GFP transgenic mice, we show for first time that Nestin(+) cells inducing the maturation of tumor vessels do not originate from the BM but presumably reside within the adventitia of adult blood vessels. Complementary ex vivo experiments using explants of murine aortas revealed that Nestin(+) multipotent stem cells (MPSCs) are mobilized from their niche and differentiated into pericytes and SMC through the influence of tumor-cell-secreted factors. We conclude that tissue-resident Nestin(+) cells are more relevant than BM-derived cells for vessel stabilization and therefore have to be considered in future strategies for anti-angiogenic therapy. The identification of proteins mediating recruitment or differentiation of local Nestin(+) cells with potential stem cell character to angiogenic blood vessels may allow the definition of new therapeutic targets to reduce tumor resistance against anti-angiogenic drugs.