Cerebrovascular disease is the third most common cause of death in developed countries, but our understanding of the cells that compose the cerebral vasculature is limited. Here, using vascular single-cell transcriptomics, we provide molecular definitions for the principal types of blood vascular and vessel-associated cells in the adult mouse brain. We uncover the transcriptional basis of the gradual phenotypic change (zonation) along the arteriovenous axis and reveal unexpected cell type differences: a seamless continuum for endothelial cells versus a punctuated continuum for mural cells. We also provide insight into pericyte organotypicity and define a population of perivascular fibroblast-like cells that are present on all vessel types except capillaries. Our work illustrates the power of single-cell transcriptomics to decode the higher organizational principles of a tissue and may provide the initial chapter in a molecular encyclopaedia of the mammalian vasculature.

Platelet-derived growth factors (PDGFs) and their receptors (PDGFRs) have served as prototypes for growth factor and receptor tyrosine kinase function for more than 25 years. Studies of PDGFs and PDGFRs in animal development have revealed roles for PDGFR-␣ signaling in gastrulation and in the development of the cranial and cardiac neural crest, gonads, lung, intestine, skin, CNS, and skeleton. Similarly, roles for PDGFR-␤ signaling have been established in blood vessel formation and early hematopoiesis. PDGF signaling is implicated in a range of diseases. Autocrine activation of PDGF signaling pathways is involved in certain gliomas, sarcomas, and leukemias. Paracrine PDGF signaling is commonly observed in epithelial cancers, where it triggers stromal recruitment and may be involved in epithelial-mesenchymal transition, thereby affecting tumor growth, angiogenesis, invasion, and metastasis. PDGFs drive pathological mesenchymal responses in vascular disorders such as atherosclerosis, restenosis, pulmonary hypertension, and retinal diseases, as well as in fibrotic diseases, including pulmonary fibrosis, liver cirrhosis, scleroderma, glomerulosclerosis, and cardiac fibrosis. We review basic aspects of the PDGF ligands and receptors, their developmental and pathological functions, principles of their pharmacological inhibition, and results using PDGF pathway-inhibitory or stimulatory drugs in preclinical and clinical contexts.Platelet-derived growth factor (PDGF) was identified more than three decades ago as a serum growth factor for fibroblasts, smooth muscle cells (SMCs), and glia cells (Kohler and Lipton 1974;Ross et al. 1974;Westermark and Wasteson 1976). Human PDGF was originally identified as a disulfide-linked dimer of two different polypeptide chains, A and B, separable using reversed phase chromatography (Johnsson et al. 1982). The B-chain (PDGF-B) was characterized by amino acid sequencing, revealing a close homology between PDGF-B and the product of the retroviral oncogene v-sis of simian sarcoma virus (SSV) (Doolittle et al. 1983;Waterfield et al. 1983). Subsequent studies confirmed that the human cellular counterpart (c-sis) was identical to PDGF-B and that autocrine PDGF activity was sufficient for SSV transformation in vitro. This was a paradigm-shifting discovery about the relationship between neoplastic cell transformation and normal growth control. For the first time, the importance of autocrine growth stimulation in neoplastic transformation was demonstrated. As discussed below, it is now well established that autocrine PDGF stimulation plays a role also in some human cancers.PDGF-A was characterized by cDNA cloning ). This resolved a paradoxical lack of correlation between secretion of PDGF-like growth factors from tumor cell lines and their expression of c-sis; it turned out that most such cell lines express PDGF-A and secrete PDGF-AA homodimers ). Together with the demonstration that PDGF-BB homodimers are produced by SSV-transformed or PDGF-Bexpressing cells, these results showed that the PDGF...

LRP1 in the NVU to promote tPA-mediated activation of PDGF-CC. Mac-1-deficient mice (Mac-1 −/−) are protected from tPA-induced BBB permeability but not from perme-ability induced by intracerebroventricular injection of active PDGF-CC. Immunofluorescence analysis demonstrates that Mac-1, LRP1, and the PDGFRα all localize to the NVU of arterioles, and following middle cerebral artery occlusion (MCAO) Mac-1 −/− mice show significantly less PDGFRα phosphorylation, BBB permeability, and infarct volume compared to wild-type mice. Bone-marrow transplantation studies indicate that resident CD11b + cells, but not bone-marrow-derived leukocytes, mediate the early activation of PDGF-CC by tPA after MCAO. Finally, using a model of thrombotic stroke with late thrombolysis, we show that wild-type mice have an increased incidence of spontaneous ICH following thrombolysis with tPA 5 h after MCAO, whereas Mac-1 −/− mice are resistant to the development of ICH even with late tPA treatment. Together, these results indicate that Mac-1 and LRP1 act as co-factors for the activation of PDGF-CC by tPA in the NVU, and suggest a novel mechanism for tightly regulating PDGFRα signaling in the NVU and controlling BBB permeability. Abstract Treatment of acute ischemic stroke with the thrombolytic tissue plasminogen activator (tPA) can significantly improve neurological outcomes; however, throm-bolytic therapy is associated with an increased risk of intra-cerebral hemorrhage (ICH). Previously, we demonstrated that during stroke tPA acting on the parenchymal side of the neurovascular unit (NVU) can increase blood-brain barrier (BBB) permeability and ICH through activation of latent platelet-derived growth factor-CC (PDGF-CC) and signaling by the PDGF receptor-α (PDGFRα). However , in vitro, activation of PDGF-CC by tPA is very inefficient and the mechanism of PDGF-CC activation in the NVU is not known. Here, we show that the integrin Mac-1, expressed on brain microglia/macrophages (denoted micro-glia throughout), acts together with the endocytic receptor Enming Joseph Su and Chunzhang Cao contributed equally to this work. Daniel A. Lawrence and Li Zhang contributed equally to this work. Electronic supplementary material The online version of this article (

Vascular diseases are major causes of death, yet our understanding of the cellular constituents of blood vessels, including how differences in their gene expression profiles create diversity in vascular structure and function, is limited. In this paper, we describe a single-cell RNA sequencing (scRNA-seq) dataset that defines vascular and vessel-associated cell types and subtypes in mouse brain and lung. The dataset contains 3,436 single cell transcriptomes from mouse brain, which formed 15 distinct clusters corresponding to cell (sub)types, and another 1,504 single cell transcriptomes from mouse lung, which formed 17 cell clusters. In order to allow user-friendly access to our data, we constructed a searchable database (http://betsholtzlab.org/VascularSingleCells/database.html). Our dataset constitutes a comprehensive molecular atlas of vascular and vessel-associated cell types in the mouse brain and lung, and as such provides a strong foundation for future studies of vascular development and diseases.

Cancer results from the concerted performance of malignant cells and stromal cells. Cell types populating the microenvironment are enlisted by the tumor to secrete a host of growth-promoting cues, thus upholding tumor initiation and progression. Platelet-derived growth factors (PDGF) support the formation of a prominent tumor stromal compartment by as of yet unidentified molecular effectors. Whereas PDGF-CC induces fibroblast reactivity and fibrosis in a range of tissues, little is known about the function of PDGF-CC in shaping the tumor-stroma interplay. Herein, we present evidence for a paracrine signaling network involving PDGF-CC and PDGF receptor-A in malignant melanoma. Expression of PDGFC in a mouse model accelerated tumor growth through recruitment and activation of different subsets of cancer-associated fibroblasts. In seeking the molecular identity of the supporting factors provided by cancer-associated fibroblasts, we made use of antibody arrays and an in vivo coinjection model to identify osteopontin as the effector of the augmented tumor growth induced by PDGF-CC. In conclusion, we establish paracrine signaling by PDGF-CC as a potential drug target to reduce stromal support in malignant melanoma. [Cancer Res 2009;69(1):369-78]

Summary Tissue growth and maintenance requires stem cell populations that self-renew, proliferate and differentiate. Maintenance of white adipose tissue (WAT) requires the proliferation and differentiation of adipocyte stem cells (ASCs) to form postmitotic, lipid-filled mature adipocytes. Here, we use the dynamic adipogenic program that occurs during hair growth to uncover an unrecognized regulator of ASC self-renewal and proliferation, Pdgfa, which activates Akt signaling to drive and maintain the adipogenic program in the skin. Pdgfa expression is reduced in aged ASCs and is required for ASC proliferation and maintenance in the dermis but not in other WATs. Our molecular and genetic studies uncover PI3K/Akt2 as a direct Pdgfa target, activated in ASCs during WAT hyperplasia and functionally required for dermal ASC proliferation. Our data therefore reveal active mechanisms that regulate ASC self-renewal in the skin and show that distinct regulatory mechanisms operate in different WAT depots.

The development of neuronal networks in the brain requires the differentiation of functional synapses. Neurobeachin (Nbea) was identified as a putative regulator of membrane protein trafficking associated with tubulovesicular endomembranes and postsynaptic plasma membranes. Nbea is essential for evoked transmission at neuromuscular junctions, but its role in the central nervous system has not been characterized. Here, we have studied central synapses of a newly generated gene-trap knockout (KO) mouse line at embryonic day 18, because null-mutant mice are paralysed and die perinatally. Although the overall brain architecture was normal, we identified major abnormalities of synaptic function in mutant animals. In acute slices from the brainstem, both spontaneous excitatory and inhibitory postsynaptic currents were clearly reduced and failure rates of evoked inhibitory responses were markedly increased. In addition, the frequency of miniature excitatory and both the frequency and amplitudes of miniature inhibitory postsynaptic currents were severely diminished in KO mice, indicating a perturbation of both action potential-dependent and -independent transmitter release. Moreover, Nbea appears to be important for the formation and composition of central synapses because the area density of mature asymmetric contacts in the fetal brainstem was reduced to 30% of wild-type levels, and the expression levels of a subset of synaptic marker proteins were smaller than in littermate controls. Our data demonstrate for the first time a function of Nbea at central synapses that may be based on its presumed role in targeting membrane proteins to synaptic contacts, and are consistent with the 'excitatory-inhibitory imbalance' model of autism where Nbea gene rearrangements have been detected in some patients.

SummaryThe role of the neurovascular niche in CNS myelin regeneration is incompletely understood. Here, we show that, upon demyelination, CNS-resident pericytes (PCs) proliferate, and parenchymal non-vessel-associated PC-like cells (PLCs) rapidly develop. During remyelination, mature oligodendrocytes were found in close proximity to PCs. In Pdgfbret/ret mice, which have reduced PC numbers, oligodendrocyte progenitor cell (OPC) differentiation was delayed, although remyelination proceeded to completion. PC-conditioned medium accelerated and enhanced OPC differentiation in vitro and increased the rate of remyelination in an ex vivo cerebellar slice model of demyelination. We identified Lama2 as a PC-derived factor that promotes OPC differentiation. Thus, the functional role of PCs is not restricted to vascular homeostasis but includes the modulation of adult CNS progenitor cells involved in regeneration.