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Complex multicellular life in mammals relies on functional cooperation of different organs for the survival of the whole organism. The kidneys play a critical part in this process through the maintenance of fluid volume and composition homeostasis, which enables other organs to fulfil their tasks. The renal endothelium exhibits phenotypic and molecular traits that distinguish it from endothelia of other organs. Moreover, the adult kidney vasculature comprises diverse populations of mostly quiescent, but not metabolically inactive, endothelial cells (ECs) that reside within the kidney glomeruli, cortex and medulla. Each of these populations supports specific functions, for example, in the filtration of blood plasma, the reabsorption and secretion of water and solutes, and the concentration of urine. Transcriptional profiling of these diverse EC populations suggests they have adapted to local microenvironmental conditions (hypoxia, shear stress, hyperosmolarity), enabling them to support kidney functions. Exposure of ECs to microenvironment-derived angiogenic factors affects their metabolism, and sustains kidney development and homeostasis, whereas EC-derived angiocrine factors preserve distinct microenvironment niches. In the context of kidney disease, renal ECs show alteration in their metabolism and phenotype in response to pathological changes in the local microenvironment, further promoting kidney dysfunction. Understanding the diversity and specialization of kidney ECs could provide new avenues for the treatment of kidney diseases and kidney regeneration.

The response of endothelial cells to signaling stimulation is critical for vascular morphogenesis, homeostasis and function. Vascular endothelial growth factor-a (VEGFA) has been commonly recognized as a pro-angiogenic factor in vertebrate developmental, physiological and pathological conditions for decades. Here we report a novel finding that genetic ablation of CDP-diacylglycerol synthetase-2 (CDS2), a metabolic enzyme that controls phosphoinositide recycling, switches the output of VEGFA signaling from promoting angiogenesis to unexpectedly inducing vessel regression. Live imaging analysis uncovered the presence of reverse migration of the angiogenic endothelium in cds2mutant zebrafish upon VEGFA stimulation, and endothelium regression also occurred in postnatal retina and implanted tumor models in mice. In tumor models, CDS2 deficiency enhanced the level of tumor-secreted VEGFA, which in-turn trapped tumors into a VEGFA-induced vessel regression situation, leading to suppression of tumor growth. Mechanistically, VEGFA stimulation reduced phosphatidylinositol (4,5)-bisphosphate (PIP2) availability in the absence of CDS2-controlled-phosphoinositide metabolism, subsequently causing phosphatidylinositol (3,4,5)-triphosphate (PIP3) deficiency and FOXO1 activation to trigger regression of CDS2-null endothelium. Thus, our data indicate that the effect of VEGFA on vasculature is context-dependent and can be converted from angiogenesis to vascular regression.

Glutamine synthetase, encoded by the gene GLUL, is an enzyme that converts glutamate and ammonia to glutamine. It is expressed by endothelial cells, but surprisingly shows negligible glutamine-synthesizing activity in these cells at physiological glutamine levels. Here we show in mice that genetic deletion of Glulin endothelial cells impairs vessel sprouting during vascular development, whereas pharmacological blockade of glutamine synthetase suppresses angiogenesis in ocular and inflammatory skin disease while only minimally affecting healthy adult quiescent endothelial cells. This relies on the inhibition of endothelial cell migration but not proliferation. Mechanistically we show that in human umbilical vein endothelial cells GLULknockdown reduces membrane localization and activation of the GTPase RHOJ while activating other Rho GTPases and Rho kinase, thereby inducing actin stress fibres and impeding endothelial cell motility. Inhibition of Rho kinase rescues the defect in endothelial cell migration that is induced by GLULknockdown. Notably, glutamine synthetase palmitoylates itself and interacts with RHOJ to sustain RHOJ palmitoylation, membrane localization and activation. These findings reveal that, in addition to the known formation of glutamine, the enzyme glutamine synthetase shows unknown activity in endothelial cell migration during pathological angiogenesis through RHOJ palmitoylation. The enzyme glutamine synthetase is active in endothelial cell migration during angiogenesis, through autopalmitoylation and the regulation of RHOJ signalling.

Transgenic adenocarcinoma of the mouse prostate (TRAMP) mice is a widely used transgenic animal model of prostate cancer (PCa). We performed a label-free quantitative proteomics analysis combined with a bioinformatics analysis on the entire prostate protein extraction from TRAMP mice and compared it with WT littermates. From 2379 total identified proteins, we presented a modest mice prostate reference proteome containing 919 proteins. 61 proteins presented a significant expression difference between two groups. The integrative bioinformatics analysis predicted the overexpression of platelet-derived growth factor B (PDGF-B) in tumor tissues and supports the hypothesis of the PDGF-B signaling network as a key upstream regulator in PCa progression. Furthermore, we demonstrated that Crenolanib, a novel PDGF receptor inhibitor, inhibited PCa cell proliferation in a dose-dependent manner. Finally, we revealed the importance of PDGF-B regulatory network in PCa progression, which will assist us in understanding the role and mechanisms of PDGF-B in promoting cancer growth and provide valuable knowledge for future research on anti-PDGF therapy.

In the middle and late stages of a gas well, the plunger gas lift process is an important means of drainage and gas production. We studied the liquid-drainage efficacy of different types of plungers (cylindrical plunger, brush plunger, and gasketed plunger) under different gas volumes and sealing differences. During the actual application in the field, the plunger has its specific size, on this foundation, a three-dimensional flow field model was created based on the actual tubing and plunger sizes, and the fluid loss and liquid-drainage process of various types of plungers was numerically simulated using the volume of fluid computational fluid dynamics numerical simulation method. The sealing mechanism of various types of plungers, as well as the impact of different gap widths between the plunger and the tubing wall on the plunger's sealing performance, were investigated. The gasketed plunger has the best sealing performance, followed by the brush plunger, and the cylindrical plunger has the worst sealing performance. The minimum gap width between the plunger and the tubing wall is an important factor affecting the plunger sealing performance; the smaller the gap width, the better the plunger's sealing performance. The experimental results show that the sealing performance is not the only condition for a reasonable plunger selection, and the size of the gas flow is also an important factor affecting the plunger's drainage. When the gas flow rate is small, the brush plunger has the best liquid-drainage efficacy, whereas the gasketed plunger has the best liquid-drainage efficacy under high gas flow.

The retinal pigment epithelium (RPE) between retinal photoreceptors and choroidal capillaries is a single layer of cells that are of critical importance to the eye. RPE cells are derived from the anterior neural plate of neuroectodermal origin. Instructed by specific molecules and signaling pathways, the RPE undergoes formation and maturation to form a functional unit together with photoreceptors. The RPE plays crucial roles in maintaining normal retinal structure and functions, such as phagocytosis; barrier function; transportation of nutrients, ions, and water; resistance to oxidative damage; maintenance of visual cycle; and production of various important factors. RPE cells have an efficient metabolic machinery to provide sufficient energy to the retina. RPE dysfunction or atrophy can lead to many retinopathies, such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Here, we discuss RPE development, functions, and roles in various ocular diseases, and the mechanisms involved. A better understanding of the functions of the RPE and related regulatory pathways may help identify novel or better therapies for the treatment of many blinding diseases.

The repair and regeneration of tissues using endogenous stem cells represents an ultimate goal in regenerative medicine. To our knowledge, human lens regeneration has not yet been demonstrated. Currently, the only treatment for cataracts, the leading cause of blindness worldwide, is to extract the cataractous lens and implant an artificial intraocular lens. However, this procedure poses notable risks of complications. Here we isolate lens epithelial stem/progenitor cells (LECs) in mammals and show that Pax6 and Bmi1 are required for LEC renewal. We design a surgical method of cataract removal that preserves endogenous LECs and achieves functional lens regeneration in rabbits and macaques, as well as in human infants with cataracts. Our method differs conceptually from current practice, as it preserves endogenous LECs and their natural environment maximally, and regenerates lenses with visual function. Our approach demonstrates a novel treatment strategy for cataracts and provides a new paradigm for tissue regeneration using endogenous stem cells.

After tissue injury, both wound sealing and apoptosis contribute to restoration of tissue integrity and functionality. Although the role of platelets (PLTs) for wound closure and induction of regenerative processes is well established, the knowledge about their contribution to apoptosis is incomplete. Here, we show that PLTs present the death receptor Fas ligand (FasL) on their surface after activation. Activated PLTs as well as the isolated membrane fraction of activated PLTs but not of resting PLTs induced apoptosis in a dose-dependent manner in primary murine neuronal cells, human neuroblastoma cells, and mouse embryonic fibroblasts. Membrane protein from PLTs lacking membrane-bound FasL (FasL△m/△m) failed to induce apoptosis. Bax/Bak-mediated mitochondrial apoptosis signaling in target cells was not required for PLT-induced cell death, but increased the apoptotic response to PLT-induced Fas signaling. In vivo, PLT depletion significantly reduced apoptosis in a stroke model and an inflammation-independent model of N-methyl-d-aspartic acid-induced retinal apoptosis. Furthermore, experiments using PLT-specific PF4Cre+ FasLfl/fl mice demonstrated a role of PLT-derived FasL for tissue apoptosis. Because apoptosis secondary to injury prevents inflammation, our findings describe a novel mechanism on how PLTs contribute to tissue homeostasis.

After tissue injury, both wound sealing and apoptosis contribute to restoration of tissue integrity and functionality. Although the role of platelets (PLTs) for wound closure and induction of regenerative processes is well established, the knowledge about their contribution to apoptosis is incomplete. Here, we show that PLTs present the death receptor Fas ligand (FasL) on their surface after activation. Activated PLTs as well as the isolated membrane fraction of activated PLTs but not of resting PLTs induced apoptosis in a dose-dependent manner in primary murine neuronal cells, human neuroblastoma cells, and mouse embryonic fibroblasts. Membrane protein from PLTs lacking membrane-bound FasL (FasL△m/△m) failed to induce apoptosis. Bax/Bak-mediated mitochondrial apoptosis signaling in target cells was not required for PLT-induced cell death, but increased the apoptotic response to PLT-induced Fas signaling. In vivo, PLT depletion significantly reduced apoptosis in a stroke model and an inflammation-independent model of N-methyl-d-aspartic acid-induced retinal apoptosis. Furthermore, experiments using PLT-specific PF4Cre+FasLfl/flmice demonstrated a role of PLT-derived FasL for tissue apoptosis. Because apoptosis secondary to injury prevents inflammation, our findings describe a novel mechanism on how PLTs contribute to tissue homeostasis.

The p53 upregulated modulator of apoptosis (PUMA) is known as an essential apoptosis inducer. Here, we report the seemingly paradoxical finding that PUMA is a proangiogenic factor critically required for the proliferation and survival of vascular and microglia cells. Strikingly, Pumadeficiency by genetic deletion or small hairpin RNA knockdown inhibited developmental and pathological angiogenesis and reduced microglia numbers in vivo, whereas Pumagene delivery increased angiogenesis and cell survival. Mechanistically, we revealed that PUMA plays a critical role in regulating autophagy by modulating Erk activation and intracellular calcium level. Our findings revealed an unexpected function of PUMA in promoting angiogenesis and warrant more careful investigations into the therapeutic potential of PUMA in treating cancer and degenerative diseases.

Formation of new vessels during development and in the mature mammal generally proceeds through angiogenesis. Although a variety of molecules and signaling pathways are known to underlie endothelial cell sprouting and remodeling during angiogenesis, many aspects of this complex process remain unexplained. Here we show that the transmembrane semaphorin6A (Sema6A) is expressed in endothelial cells, and regulates endothelial cell survival and growth by modulating the expression and signaling of VEGFR2, which is known to maintain endothelial cell viability by autocrine VEGFR signaling. The silencing of Sema6A in primary endothelial cells promotes cell death that is not rescued by exogenous VEGF-A or FGF2, attributable to the loss of prosurvival signaling from endogenous VEGF. Analyses of mouse tissues demonstrate that Sema6A is expressed in angiogenic and remodeling vessels. Mice with null mutations of Sema6A exhibit significant defects in hyaloid vessels complexity associated with increased endothelial cell death, and in retinal vessels development that is abnormally reduced. Adult Sema6A-null mice exhibit reduced tumor, matrigel, and choroidal angiogenesis compared with controls. Sema6A plays important roles in development of the nervous system. Here we show that it also regulates vascular development and adult angiogenesis.

Formation of new vessels during development and in the mature mammal generally proceeds through angiogenesis. Although a variety of molecules and signaling pathways are known to underlie endothelial cell sprouting and remodeling during angiogenesis, many aspects of this complex process remain unexplained. Here we show that the transmembrane semaphorin6A (Sema6A) is expressed in endothelial cells, and regulates endothelial cell survival and growth by modulating the expression and signaling of VEGFR2, which is known to maintain endothelial cell viability by autocrine VEGFR signaling. The silencing of Sema6Ain primary endothelial cells promotes cell death that is not rescued by exogenous VEGF-A or FGF2, attributable to the loss of prosurvival signaling from endogenous VEGF. Analyses of mouse tissues demonstrate that Sema6A is expressed in angiogenic and remodeling vessels. Mice with null mutations of Sema6Aexhibit significant defects in hyaloid vessels complexity associated with increased endothelial cell death, and in retinal vessels development that is abnormally reduced. Adult Sema6A-null mice exhibit reduced tumor, matrigel, and choroidal angiogenesis compared with controls. Sema6A plays important roles in development of the nervous system. Here we show that it also regulates vascular development and adult angiogenesis.

Beyond its role in immunity, complement mediates a wide range of functions in the context of morphogenetic or tissue remodeling processes. Angiogenesis is crucial during tissue remodeling in multiple pathologies; however, the knowledge about the regulation of neovascularization by the complement components is scarce. Here we studied the involvement of complement in pathological angiogenesis. Strikingly, we found that mice deficient in the central complement component C3 displayed increased neovascularization in the model of retinopathy of prematurity (ROP) and in the in vivo Matrigel plug assay. In addition, antibody-mediated blockade of C5, treatment with C5aR antagonist, or C5aR deficiency in mice resulted in enhanced pathological retina angiogenesis. While complement did not directly affect angiogenesis-related endothelial cell functions, we found that macrophages mediated the antiangiogenic activity of complement. In particular, C5a-stimulated macrophages were polarized toward an angiogenesis-inhibitory phenotype, including the up-regulated secretion of the antiangiogenic soluble vascular endothelial growth factor receptor-1. Consistently, macrophage depletion in vivo reversed the increased neovascularization associated with C3- or C5aR deficiency. Taken together, complement and in particular the C5a-C5aR axes are potent inhibitors of angiogenesis.

Beyond its role in immunity, complement mediates a wide range of functions in the context of morphogenetic or tissue remodeling processes. Angiogenesis is crucial during tissue remodeling in multiple pathologies; however, the knowledge about the regulation of neovascularization by the complement components is scarce. Here we studied the involvement of complement in pathological angiogenesis. Strikingly, we found that mice deficient in the central complement component C3 displayed increased neovascularization in the model of retinopathy of prematurity (ROP) and in the in vivo Matrigel plug assay. In addition, antibody-mediated blockade of C5, treatment with C5aR antagonist, or C5aR deficiency in mice resulted in enhanced pathological retina angiogenesis. While complement did not directly affect angiogenesis-related endothelial cell functions, we found that macrophages mediated the antiangiogenic activity of complement. In particular, C5a-stimulated macrophages were polarized toward an angiogenesis-inhibitory phenotype, including the up-regulated secretion of the antiangiogenic soluble vascular endothelial growth factor receptor-1. Consistently, macrophage depletion in vivo reversed the increased neovascularization associated with C3- or C5aR deficiency. Taken together, complement and in particular the C5a-C5aR axes are potent inhibitors of angiogenesis.

Ligand interaction with cognate cell-surface receptor often promotes receptor internalization, protecting cells from prolonged or excessive signaling from extracellular ligands. Compounds that induce internalization of surface receptors prevent ligand binding to cognate cell-surface receptors serving as inhibitors. Here, we show that synthetic polyriboguanosine (poly G) and oligo-deoxyriboguanosine (oligo G) reduce endothelial levels of surface neuropilin-1 (NRP1), a receptor shared by semaphorin 3A and vascular endothelial growth factor (VEGF), which plays critical roles in angiogenesis. Oligo G also reduces levels of cell-surface scavenger receptor expressed by endothelial cells I (SREC-I), but not levels of NRP2, gp130, CD31, VEGFR-1, or VEGFR-2. Poly or oligo A, T, and C do not promote NRP1 or SREC-I internalization. We find that oligo G binds to NRP1 with high affinity (Kd:1.3 ± 0.16nM), bridges the extracellular domain of NRP1 to that of SREC-I, and induces coordinate internalization of NRP1 and SREC-I. In vitro, oligo G blocks the binding and function of VEGF165 in endothelial cells. In vivo, intravitreal administration of oligo G reduces choroidal neovascularization in mice. These results demonstrate that synthetic oligo G is an inhibitor of pathologic angiogenenesis that reduces cell-surface levels and function of NRP1 acting as an internalization inducer.

Despite its early discovery and high sequence homology to the other VEGF family members, the biological function of VEGF-B remained debatable for a long time, and VEGF-B has received little attention from the field thus far. Recently, we and others have found that (1) VEGF-B is a potent survival factor for different types of cells by inhibiting apoptosis via suppressing the expression of BH3-only protein and other apoptotic/cell death-related genes. (2) VEGF-B has a negligible role in inducing blood vessel growth in most organs. Instead, it is critically required for blood vessel survival. VEGF-B targeting inhibited pathological angiogenesis by abolishing blood vessel survival in different animal models. (3) Using different types of neuro-injury and neurodegenerative disease models, VEGF-B treatment protected endangered neurons from apoptosis without inducing undesired blood vessel growth or permeability. Thus, VEGF-B is the first member of the VEGF family that has a potent survival/anti-apoptotic effect, while lacking a general angiogenic activity. Our work thus advocates that the major function of VEGF-B is to act as a “survival”, rather than an “angiogenic” factor, and implicates a therapeutic potential of VEGF-B in treating different types of vascular and neurodegenerative diseases.

The platelet-derived growth factors are implicated in development of fibrotic reactions and disease in several organs. We have overexpressed platelet-derived growth factor-C in the heart using the α-myosin heavy chain promoter and created a transgenic mouse that exhibits cardiac fibrosis followed by hypertrophy with sex-dependent phenotypes. The transgenic mice developed several pathological changes including cardiac fibroblast proliferation and deposition of collagen, hypertrophy, vascular defects, and the presence of Anitschkow cells in the adult myocardium. Male mice developed a hypertrophic phenotype, whereas female mice were more severely affected and developed dilated cardiomyopathy, leading to heart failure and sudden death. The vascular defects initially included dilation of microvessels and vascular leakage. Subsequently, a marked loss of microvessels, formation of large vascular sac-like structures, and an increased density of smooth muscle-coated vessels were observed in the myocardium. In part, the observed vascular changes may be because of an up-regulation of vascular endothelial growth factor in cardiac fibroblasts of the transgenic hearts. This unique animal model reveals that a potent mitogen for cardiac fibroblasts result in an expansion of the interstitium that induce a secondary sex-dependent hypertrophic response in the cardiomyocytes.

A newly discovered PDGF isoform, PDGF‐CC, is expressed in actively angiogenic tissues such as placenta, some embryonic tissues, and tumors. We test the possibility that PDGF‐CC promotes angiogenesis in vivo. The core domain (mature form) of human PDGF‐CC is sufficiently potent to stimulate neovascularization in the mouse cornea. The corneal angiogenic response induced by PDGF‐CC is robust although the area of neovascularization is smaller than those of FGF‐2‐and VEGF‐stimulated angiogenesis. Similarly, PDGF‐BB and PDGF‐AB induce angiogenic responses virtually indistinguishable from PDGF‐CCstimulated vessels. In contrast, PDGF‐AA displays only a weak angiogenic response in the mouse cornea. Although there was no significant difference in incorporation of mural cells to the newly formed blood vessels induced by PDGF‐BB and ‐CC, the percentage of mural cell positive vessels induced by PDGF‐AA was greater than those induced by FGF‐2, PDGF‐BB, and PDGF‐CC. In the developing chick embryo, PDGF‐CC induced branch sprouts from established blood vessels. In PDGF receptor‐transfected endothelial cells, PDGF‐CC activated the PDGF receptor alpha subunit (PDGFR‐a). PDGF‐CC, but not PDGF‐AA, was able to activate PDGFR‐p receptor in endothelial cells that coexpress both α and β forms of receptors. Thus, the PDGF‐CC‐mediated angiogenic response is most likely transduced by PDGF‐aa and ‐ap receptors. These data demonstrate that the PDGF family is a complex and important group of proangiogenic factors.—Cao, R., Bråkenhielm, E., Li, X., Pietras, K., Widenfalk, J, Östman, A., Eriksson, U., Cao, Y. Angiogenesis stimulated by PDGF‐CC, a novel member in the PDGF family, involves activation of PDGFR‐aa and ‐ap receptors. FASEB J. 16, 1575–1583 (2002)

Vascular endothelial growth factor B (VEGF-B), a member of the VEGF/PDGF family, is highly expressed in many tissues with two differentially spliced transcripts generating two secreted isoforms, VEGF-B167 and VEGF-B186. In this work, we have investigated the expression of VEGF-B in tissues and cell lines using techniques that can distinguish the two isoforms. The results showed that the VEGF-B167 isoform was predominantly expressed in most tissues, accounting for more than 80% of the total VEGF-B transcripts. The VEGF-B186 isoform was expressed at lower levels and only in a limited number of tissues. Moreover, the VEGF-B186 isoform was up-regulated in mouse and human tumor cell lines and primary tumors compared with their corresponding normal tissues. Taken together, our data suggest a fme genetic control of the expression of the two isoforms of VEGF-B, implying tissue- and cell-specific roles of the two VEGF-B isoforms.

Endothelial cells (ECs) harbor distinct phenotypical and functional characteristics depending on their tissue localization and contribute to brain, eye, lung, and muscle diseases such as dementia, macular degeneration, pulmonary hypertension, and sarcopenia. To study their function, isolation of pure ECs in high quantities is crucial. Here, we describe protocols for rapid and reproducible blood vessel EC purification established for scRNA sequencing from murine tissues using mechanical and enzymatic digestion followed by magnetic and fluorescence-activated cell sorting.

Endothelial cells (ECs) from the small intestine, colon, liver, and heart have distinct phenotypes and functional adaptations that are dependent on their physiological environment. Gut ECs adapt to low oxygen, heart ECs to contractile forces, and liver ECs to low flow rates. Isolating high-purity ECs in sufficient quantities is crucial to study their functions. Here, we describe protocols combining magnetic and fluorescent activated cell sorting for rapid and reproducible EC purification from four adult murine tissues.

In earlier studies we found up-regulation of promoter P3 transcription and total igf2 expression with concomitant down-regulation of h19 expression in human hepatoblastoma (HB). In this study, we investigated the methylation status of these 2 genes and their abnormal regulation in 7 hepatoblastomas and 5 normal counterpart tissues. A specific part of the P3 region of igf2 was de-methylated in tumors with up-regulation of the promoter activity. A site-specific DNA hypermethylation in the h19 5'-region was found in tumor tissues with concomitant down-regulation of this gene. Therefore, abnormal methylation was found to be correlated with altered regulation of igf2 and h19 expression in human hepatoblastoma and may be involved in the genesis of this tumor. Int. J. Cancer 75:176–180, 1998. © 1998 Wiley-Liss, Inc.

IGF2 has been shown to be expressed preferentially from the paternally derived allele, although the maternal allele can be found active during both prenatal and postnatal development as well as in neoplastic tumours in humans. We addressed here whether or not the biallelic expression patterns that can be seen during postnatal human liver development reflected a coordinated change in the activities of the four promoters of human IGF2. We show here that the P2, P3 and P4 promoters, but not the P1 promoter, display monoallelic activity in embryonic, neonatal and younger infant liver specimens. The P2, P3 and P4 promoters can, however, be found active either monoallelically or biallelically or even monoallelically on opposite parental alleles in older infant and adult liver specimens. In contrast, H19, which is closely linked to IGF2, is monoallelically expressed in all postnatal liver samples analysed. We conclude that the functional imprinting status of IGF2 during postnatal liver development appears to be promoter/enhancer-specific and either partly or completely independent of H19.

Platelet-derived growth factor C signaling promotes radiation proctopathy development and can be inhibited in a mouse model.

Endothelial cells (ECs) exhibit phenotypic and functional tissue specificities, critical for studies in the vascular field and beyond. Thus, tissue-specific methods for isolation of highly purified ECs are necessary. Kidney, spleen, and testis ECs are relevant players in health and diseases such as chronic kidney disease, acute kidney injury, myelofibrosis, and cancer. Here, we provide tailored protocols for rapid and reproducible EC purification established for scRNA sequencing from these adult murine tissues using the combination of magnetic- and fluorescence-activated cell sorting.