Pericyte Population Research Articles

Notch3 establishes brain vascular integrity by regulating pericyte number

Brain pericytes are important regulators of brain vascular integrity, permeability and blood flow. Deficiencies of brain pericytes are associated with neonatal intracranial hemorrhage in human fetuses, as well as stroke and neurodegeneration in adults. Despite the important functions of brain pericytes, the mechanisms underlying their development are not well understood and little is known about how pericyte density is regulated across the brain. The Notch signaling pathway has been implicated in pericyte development, but its exact roles remain ill defined. Here, we report an investigation of the Notch3 receptor using zebrafish as a model system. We show that zebrafish brain pericytes express notch3 and that notch3 mutant zebrafish have a deficit of brain pericytes and impaired blood-brain barrier function. Conditional loss- and gain-of-function experiments provide evidence that Notch3 signaling positively regulates brain pericyte proliferation. These findings establish a new role for Notch signaling in brain vascular development whereby Notch3 signaling promotes expansion of the brain pericyte population.

Fate tracing reveals hepatic stellate cells as dominant contributors to liver fibrosis independent of its aetiology

Although organ fibrosis causes significant morbidity and mortality in chronic diseases, the lack of detailed knowledge about specific cellular contributors mediating fibrogenesis hampers the design of effective anti-fibrotic therapies. Different cellular sources including tissue-resident and bone marrow-derived fibroblasts, pericytes and epithelial cells have been suggested to give rise to myofibroblasts, but their relative contributions remain controversial, with profound differences between organs and different diseases. Here we employ a novel Cre-transgenic mouse that marks 99% of hepatic stellate cells (HSCs), a liver-specific pericyte population, to demonstrate that HSCs give rise to 82-96% of myofibroblasts in models of toxic, cholestatic and fatty liver disease. Moreover, we exclude that HSCs function as facultative epithelial progenitor cells in the injured liver. On the basis of these findings, HSCs should be considered the primary cellular target for anti-fibrotic therapies across all types of liver disease.

New Light Is Shed on the Enigmatic Origin of the Lung Myofibroblast

New Light Is Shed on the Enigmatic Origin of the Lung Myofibroblast

Role of Lung Pericytes and Resident Fibroblasts in the Pathogenesis of Pulmonary Fibrosis

The origin of cells that make pathologic fibrillar collagen matrix in lung disease has been controversial. Recent studies suggest mesenchymal cells may contribute directly to fibrosis. To characterize discrete populations of mesenchymal cells in the normal mouse lung and to map their fate after bleomycin-induced lung injury. We mapped the fate of Foxd1-expressing embryonic progenitors and their progeny during lung development, adult homeostasis, and after fibrosing injury in Foxd1-Cre; Rs26-tdTomato-R mice. We studied collagen-I(α)1-producing cells in normal and diseased lungs using Coll-GFP(Tg) mice. Foxd1-expressing embryonic progenitors enter lung buds before 13.5 days post-conception, expand, and form an extensive lineage of mesenchymal cells that have characteristics of pericytes. A collagen-I(α)1-expressing mesenchymal population of distinct lineage is also found in adult lung, with features of a resident fibroblast. In contrast to resident fibroblasts, Foxd1 progenitor-derived pericytes are enriched in transcripts for innate immunity, vascular development, WNT signaling pathway, and cell migration. Foxd1 progenitor-derived pericytes expand after bleomycin lung injury, and activate expression of collagen-I(α)1 and the myofibroblast marker αSMA in fibrotic foci. In addition, our studies suggest a distinct lineage of collagen-I(α)1-expressing resident fibroblasts that also expands after lung injury is a second major source of myofibroblasts. We conclude that the lung contains an extensive population of Foxd1 progenitor-derived pericytes that are an important lung myofibroblast precursor population.

OP0155 A Novel Model of Systemic Sclerosis?: Loss of PTEN in Fibroblasts Results in Lung Fibrosis in A CCN2/CTGF-Dependent Fashion

BackgroundScleroderma fibroblasts are characterized by elevated adhesive signaling. PTEN, a phosphatase that suppresses adhesive signaling, expression is reduced in scleroderma dermal fibroblasts. Previously, we showed that loss of PTEN expression...

The increase of pericyte population in human neuromuscular disorders supports their role in muscle regeneration in vivo

Pericytes are periendothelial cells that have been involved in many different functions including a possible role as mesodermal stem/progenitor cells. In the present study we demonstrate that alkaline phosphatase (AP) expression is specific for human muscular pericytes and can be used as a marker to identify them in skeletal muscle biopsies. We studied the pericyte population in skeletal muscle biopsies from controls, myopathic and neuropathic patients. We observed a significant increase in the number of pericytes only in myopathies that correlated with the number of NCAM(+) fibres, suggesting that an active muscular degenerative/regenerative process is related to an increase in the pericyte population. AP(+) pericytes sorted from skeletal muscle samples were able to activate the myogenic programme and fuse with both mononucleate satellite cells and mature multinucleated myotubes in vitro, demonstrating that they could participate in muscle regeneration. In accordance, pericytes expressing the myogenic transcription factor MyoD were found in biopsies of myopathic biopsies. All these data support the hypothesis that, apart from satellite cells, pericytes may play an important role in muscle regeneration in adult human muscles in vivo.

Perivascular Stem Cells: A Prospectively Purified Mesenchymal Stem Cell Population for Bone Tissue Engineering

Adipose tissue is an ideal source of mesenchymal stem cells for bone tissue engineering: it is largely dispensable and readily accessible with minimal morbidity. However, the stromal vascular fraction (SVF) of adipose tissue is a heterogeneous cell population, which leads to unreliable bone formation. In the present study, we prospectively purified human perivascular stem cells (PSCs) from adipose tissue and compared their bone-forming capacity with that of traditionally derived SVF. PSCs are a population (sorted by fluorescence-activated cell sorting) of pericytes (CD146+CD34-CD45-) and adventitial cells (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. Here, we found that PSCs underwent osteogenic differentiation in vitro and formed bone after intramuscular implantation without the need for predifferentiation. We next sought to optimize PSCs for in vivo bone formation, adopting a demineralized bone matrix for osteoinduction and tricalcium phosphate particle formulation for protein release. Patient-matched, purified PSCs formed significantly more bone in comparison with traditionally derived SVF by all parameters. Recombinant bone morphogenetic protein 2 increased in vivo bone formation but with a massive adipogenic response. In contrast, recombinant Nel-like molecule 1 (NELL-1; a novel osteoinductive growth factor) selectively enhanced bone formation. These studies suggest that adipose-derived human PSCs are a new cell source for future efforts in skeletal regenerative medicine. Moreover, PSCs are a stem cell-based therapeutic that is readily approvable by the U.S. Food and Drug Administration, with potentially increased safety, purity, identity, potency, and efficacy. Finally, NELL-1 is a candidate growth factor able to induce human PSC osteogenesis.

The isolation and in situ identification of MSCs residing in loose connective tissues using a niche-preserving organ culture system

Mesenchymal stem cells (MSCs) have been discovered in a multitude of organs, but their distribution and identity are still uncertain. Furthermore, loose connective tissue (LCT) is dispersed throughout virtually all organs, but its biological role in tissue homeostasis is unclear. Here, we describe a unique organ culture system to explore the omnipresence and in situ identity of MSCs among the LCTs. This culture system included the use of the fibrin hydrogel coupled with dynamic culture conditions, using native LCTs obtained from various organs as starting materials. This culture allowed MSC outgrowth into the hydrogel to be robustly supported, while maintaining the structural integrity of LCTs during in vitro culture. Subcultured outgrown cells fulfilled the minimal requirements for defining MSCs on the basis of clonogenicity, multipotency, and immunophenotypic characteristics. In vitro label-retaining assay demonstrated that the numbers of mobilized and proliferated cells in situ increased in the pericapillary region and expressed both MSCs and pericytes markers, indicating that the in situ identity of MSCs represents a certain population of pericapillary pericytes. Our results indicate that this culture system affords a unique strategy for both isolating MSCs and recapitulating their niche in LCTs.

Proteome of human subcutaneous adipose tissue stromal vascular fraction cells versus mature adipocytes based on DIGE.

Adipose tissue contains a heterogeneous population of mature adipocytes, endothelial cells, immune cells, pericytes, and preadipocytic stromal/stem cells. To date, a majority of proteomic analyses have focused on intact adipose tissue or isolated adipose stromal/stem cells in vitro. In this study, human subcutaneous adipose tissue from multiple depots (arm and abdomen) obtained from female donors was separated into populations of stromal vascular fraction cells and mature adipocytes. Out of 960 features detected by 2-D gel electrophoresis, a total of 200 features displayed a 2-fold up- or down-regulation relative to each cell population. The protein identity of 136 features was determined. Immunoblot analyses comparing SVF relative to adipocytes confirmed that carbonic anhydrase II was up-regulated in both adipose depots while catalase was up-regulated in the arm only. Bioinformatic analyses of the data set determined that cytoskeletal, glycogenic, glycolytic, lipid metabolic, and oxidative stress related pathways were highly represented as differentially regulated between the mature adipocytes and stromal vascular fraction cells. These findings extend previous reports in the literature with respect to the adipose tissue proteome and the consequences of adipogenesis. The proteins identified may have value as biomarkers for monitoring the physiology and pathology of cell populations within subcutaneous adipose depots.

Abstract 5115: Purified Human Muscle-Derived Pericytes Support Formation of Vascular Structures and Promote Angiogenesis After Myocardial Infarction

We have recently shown that perivascular cells isolated from multiple human tissues represent stem cells endowed with mesodermal lineage potentials, and the injection of multipotent muscle-derived pericytes repair damaged skeletal muscle in vivo . Our current study further demonstrated that transplantation of human muscle-derived pericytes (3.0×10 5 cells/heart) into the ischemic myocardium of NOD/SCID mice sustains the cardiac function of the infarcted hearts. Here, we tested the hypothesis that purified CD34 − /45 − /56 − /146 high pericytes support formation of structures of blood vessels and promote angiogenesis post infarction. To illustrate the potential vessel-forming properties of purified pericytes, pericytes and endothelial cells were co-cultured in Matrigel in vitro, and co-formation of capillary-like structures was observed. To evaluate the angiogenic effect of pericytes in vivo , histological examination was performed two weeks after transplantation. Capillary density was measured by anti-CD31 staining on tissue sections. Capillary density within peri-infarct areas was 43% higher in the pericyte-injected group than in the saline-injected group ( P <0.05). Myocardial fibrosis estimated by Masson’s trichrome staining revealed a 38% reduction of the scar area in pericyte-injected hearts when compared to saline-injected hearts. These findings indicated that transplantation of pericytes sustains the cardiac function of the infarcted left ventricle presumably through enhancement of angiogenesis and reduction of myocardial fibrosis. We further examined potential mediators of pericyte-induced cardiac repair in the ischemic micro-environment by culturing pericytes under hypoxic conditions (2.5% oxygen) in vitro for 24 hours. Real-time quantitative PCR analysis showed that vascular endothelial growth factor (VEGF)-related genes, including VEGF 165 and VEGF receptor 1 and 2, were significantly up-regulated in the pericyte population. Overall, the data suggested a role of VEGF-regulated angiogenesis on pericyte-mediated cardiac repair. Experiments are being performed to elucidate the potential of pericytes to differentiate into cardiac cell lineages and explore the regulatory mechanisms involved in vivo .

The Cochlear Pericytes

Cochlear pericytes are not well characterized. The aim of this study was to further advance the characterization of cochlear pericyte location and distribution, with particular focus on pericyte-related proteins on the capillaries of the cochlear lateral wall that are functionally integral to structure, contraction, and gap junction transport. Cochlear pericytes were identified by the immunofluorescence labeling of pericyte marker proteins, including alpha-smooth muscle actin (alpha-SMA), desmin, Thy-1, tropomyosin, and NG2, and by morphological identification, using fluorescence, electron, and differential interference contrast microscopy. Pericytes were predominately found in the capillary network of the cochlear lateral wall, with considerable morphological heterogeneity across different types of microvessels. For example, pericytes on the vessels of the spiral ligament (V/SL) strongly expressed a gap junction protein, connexin 40, and were positive for alpha-SMA, tropomyosin, and desmin. In contrast, pericytes on the vessels of the stria vascularis (V/SV) were positive for desmin, and were negative for alpha-SMA and tropomyosin. The capillary networks of the cochlear lateral wall comprise a rich population of pericytes. These pericytes are morphologically heterogeneous, with protein expression potentially indicative of function.

Platelet-Derived Growth Factor Signaling Through Ephrin-B2 Regulates Hepatic Vascular Structure and Function

Cirrhosis is associated with prominent changes in sinusoidal structure and function. Although the resident pericyte in liver, the hepatic stellate cell (HSC), is well characterized in the process of fibrogenesis, signaling pathways that regulate HSC vascular function are less developed. Because pericyte populations outside the liver are increasingly being recognized as a key cell type for angiogenesis and changes in vascular structure, in this study, we explore new HSC-signaling pathways that regulate sinusoidal structure and function. Real-time video microscopy and quantitative software analysis of vascular tube formation were used to measure HSC angiogenesis in vitro. Platelet-derived growth factor (PDGF) and ephrin-signaling pathways were modulated using molecular and pharmacologic techniques. Complementary whole animal studies were performed to correlate in vitro findings with pericyte functions in vivo. We show that PDGF promotes a phenotype of HSC evidenced by enhanced HSC-driven vascular tube formation in vitro and enhanced HSC coverage of sinusoids in vivo. This angiogenic phenotype modulates specific pericyte vascular functions including permeability and pressure regulation. Furthermore, we identify a key role for ephrin-B2 as a downstream effector of PDGF signaling. These studies elucidate novel HSC-signaling pathways that regulate microvascular structure and function in liver.

Identification of fibroblast heterogeneity in the tumor microenvironment

Tumors are unorganized organs that contain many different cell types. In the recent years, many studies have reported that primary tumors contain fibroblasts/myofibroblasts (carcinoma-associated fibroblasts), mesenchymal cells such as pericytes/mural cells and other vascular smooth muscle cells. Several different markers are used routinely to identify carcinoma-associated fibroblasts (CAFs) such as alpha-smooth muscle actin (α-SMA), vimentin, S100A4 protein/fibroblast specific protein-1 (FSP1) and type I collagen. Likewise markers such as platelet derived growth factor receptor-beta (PDGFRβ) and NG2 chondroitin sulfate proteoglycan (NG2) are used to identify mesenchymal cells such as pericytes and other vasculature associated smooth muscle cells. It is still unknown whether these markers overlap with each other or identify a unique population of cells within the tumor microenvironment. Therefore in the present study we utilized two different mouse models of cancer, the Rip1Tag2 mice that develop progressive pancreatic cancer and an orthotopic 4T1 breast cancer model, to study the overlap between six different mesenchymal markers commonly used in mouse cancer research. Our study demonstrates that among all the markers, S100A4/FSP1 identifies a unique population of fibroblasts with minimal overlap with markers for αSMA, PDGFRβ and NG2. Vimentin and type I collagen are not specific markers for fibroblasts in these tumors. αSMA, PDGFRβ and NG2 significantly overlap with each other in identifying a mixed population of fibroblasts (activated or resting), myofibroblasts, pericytes and vascular smooth muscle cells. Collectively, this study demonstrates that tumor microenvironment associated fibroblasts are a heterogeneous population and thus, the use of αSMA or vimentin as the only markers will not identify all the CAFs.

Feline Systemic Reactive Angioendotheliomatosis: Eight Cases and Literature Review

A rare, multisystemic intravascular proliferative disorder was identified postmortem in eight cats. The majority of these cats died or were euthanized following episodes of dyspnea, lethargy, and anorexia. Microscopic examination revealed occlusive, intraluminal proliferations of spindle cells within small vessels. The heart was consistently involved, and myocardial dysfunction was the probable cause of illness in all cats. Immunohistochemically, the majority of intravascular cells expressed von Willebrand factor, and a smaller number expressed smooth muscle actin, compatible with a dual population of endothelial cells and pericytes, suggesting a reactive rather than a neoplastic process. Four cases of a similar feline vascular disorder from the veterinary literature are reviewed. The histopathology resembles reactive angioendotheliomatosis in humans, a benign cutaneous intravascular endothelial and pericytic proliferative condition. However, in contrast, this feline disease is multisystemic and fatal. We propose the name "feline systemic reactive angioendotheliomatosis" for this unique, idiopathic disorder of domestic cats.

Quantification of rat retinal growth and vascular population changes after single and split doses of proton irradiation: translational study using stereology methods.

This study quantified architectural and population changes in the rat retinal vasculature after proton irradiation using stereology. A 100 MeV conformal proton beam delivered 8, 14, 20 and 28 Gy as single and split doses to the whole eye. The vascular networks were prepared from retinal digests. Stereological methods were used to obtain the area of the retina and unbiased estimates of microvessel/artery/vein endothelial, pericyte and smooth muscle population, and vessel length. The retinal area increased progressively in the unirradiated, age-matched controls and in the retinas irradiated with 8 and 14 Gy, indicating uniform progressive retinal growth. No growth occurred after 20 and 28 Gy. Regression analysis of total endothelial cell number in all vessels (arteries, veins and capillaries) after irradiation documented a progressive time- and dose-dependent cell loss occurring over 15 to 24 months. The difference from controls was significant (P<0.01) after 28 Gy given in single and split doses and after 20 Gy given as a split dose (P<0.05). Total vessel length in microvessel was significantly shortened at 20 and 28 Gy compared to that of controls (P<0.05). No evident dose recovery was observed in the endothelial populations after split doses. At 10 Gy, the rate of endothelial cell loss, a dose parameter used to characterize the time- and dose-dependent loss of the endothelial population, was doubled.

Dose response of rat retinal microvessels to proton dose schedules used clinically: a pilot study

Purpose: This preclinical rat pilot study quantifies retinal microvessel, endothelial, and pericyte population changes produced by proton irradiation Methods and Materials: The left eyes of rats were irradiated with single doses of 8, 14, 20, and 28 Gy protons; right eyes, with two fractions. Animals were euthanized, and eyes were removed; elastase digests were prepared, and cell populations were counted in sample fields. Results were compared with unirradiated controls. Results: Progressive time- and dose-dependent endothelial cell loss occurred following all schedules. Cell loss was significantly different from control values ( p < 0.001) following 28 Gy and following 20 Gy ( p < 0.05) in a single dose. Endothelial cell loss was the same for single- and split-dose schedules. Progressive endothelial cell loss produced vessel collapse and acellular vessel strands. Endothelial cells were in the G 0 phase of the mitotic cycle. 28 Gy produced photoreceptor cell loss. Conclusion: The retinal digest is an elegant bioassay to quantify the microvessel population response. Single- and split-dose schedules appear to yield similar outcomes, in terms of endothelial cell density.

Diversity within pericytes.

1. Pericytes are cells of microvessels (arterioles, capillaries and venules) that wrap around endothelial cells. They are most abundant on venules and are common on capillaries. 2. The pericyte population is highly variable between different tissues and organs, probably in a manner reflecting postarteriolar hydrostatic pressures. Pericytes are more abundant in the distal legs and feet, again suggesting a hydrostatic pressure-driven mechanical role for pericytes as protectors of microvessel wall integrity. 3. Pericyte alteration or degeneration is linked directly with microangiopathy in diabetes, scleroderma, hypertension, dementias and, possibly, inappropriate calcification of blood vessels. 4. Pericytes are functionally codependent on endothelial cells. Each cell type influences each others' mitotic rate and probably phenotypic expression. 5. Pericytes are not randomly located around microvessels. Instead, they are located adjacent to or over endothelial cell junctions of venules and especially over gaps between endothelial cells during inflammation. Pericytes are emerging as essential components of the microvessel wall, with metabolic, signalling and mechanical roles to support the endothelial cell.

Comparison of image analysis and stereologic techniques used to quantify endothelial cell and pericyte populations in the rat retinal microvessels following proton irradiation: Results of a pilot study

Comparison of image analysis and stereologic techniques used to quantify endothelial cell and pericyte populations in the rat retinal microvessels following proton irradiation: Results of a pilot study

The role of pericytes in controlling angiogenesis in vivo.

In order to evaluate the interaction between endothelial cells and the perivascular pericytes during physiological angiogenesis, stereological analysis of fine structure was performed on samples of rat skeletal muscle where capillary growth was induced to a similar extent by three different interventions (indirect electrical stimulation, vasodilatation by alpha 1-blockade, stretch due to synergist extirpation). There was a significant reduction in the relative area of contact between pericytes and the capillary abluminal surface with stimulation, and withdrawal of pericyte processes coincided with an increase in anatomical capillary supply. These data indicate that pericytes may play an anti-angiogenic role in vivo in normal adult tissue similar to that proposed for in vitro models of angiogenesis, with their retraction during increased muscle activity possibly releasing endothelial cells from their contact inhibition. However, following long-term peripheral vasodilatation expansion of the capillary bed was accompanied by a co-ordinated increase in pericytes, such that coverage of capillaries was similar to that in control muscles. In addition, growth of capillaries following prolonged stretch resulted in a slightly greater increase in the pericyte population, suggesting they may be permissive for endothelial cell migration. Thus, the role of pericytes in controlling physiological angiogenesis is dependent on the nature of the initial stimulus, suggesting that in vitro data have to be interpreted with caution when discussing the mechanism of capillary growth in vivo.

Angiomatosis with angiokeratoma-like features in children: a light microscopic and immunophenotypic examination of four cases.

We have identified three patients with an initial clinical or biopsy diagnosis of angiokeratoma, all of whom were found to have a more extensive vascular lesion within the surgical excision. A fourth patient with identical histologic findings had no specified clinical diagnosis and his first procedure was excisional. The patients ranged in age from 7 to 16 years, and the lesions were located on the buttock, thigh, calf, and foot. Macroscopic appearances included mildly keratotic pink-red or blue-grey macules (three cases) and pink macules with focal ulceration (one case). In three of the four cases, a shave biopsy diagnosis of angiokeratoma had been made, and the extensive and deeply infiltrative nature of the vascular proliferation was recognized only at subsequent resection, at which point angiomatosis was diagnosed. In the fourth case. excisional biopsy was attempted at presentation, and the superficial morphology was angiokeratoma-like, but the vascular proliferation was present in the deep subcutaneous fat. CD31 and CD34 reactivity was present in the superficial and deep vessels in all cases, and lesional vessels were rimmed by a bland population of smooth muscle actin positive pericytes, findings that differentiate these cases from angiokeratoma, which has previously been reported to be CD34 negative. We conclude that the dilated vascular spaces that typify angiokeratoma may also be seen overlying a deep vasoformative process that is not amenable to resection, and suggest that caution should be exercised in evaluating small biopsies with angiokeratoma-like appearance.