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Cell migration is a key process in the shaping and formation of tissues. During sprouting angiogenesis, endothelial tip cells invade avascular tissues by generating actomyosin-dependent forces that drive cell migration and vascular expansion. Surprisingly, endothelial cells (ECs) can still invade if actin polymerization is inhibited. In this study, we show that endothelial tip cells employ an alternative mechanism of cell migration that is dependent on Aquaporin (Aqp)-mediated water inflow and increase in hydrostatic pressure. In the zebrafish, ECs express aqp1a.1 and aqp8a.1 in newly formed vascular sprouts in a VEGFR2-dependent manner. Aqp1a.1 and Aqp8a.1 loss-of-function studies show an impairment in intersegmental vessels formation because of a decreased capacity of tip cells to increase their cytoplasmic volume and generate membrane protrusions, leading to delayed tip cell emergence from the dorsal aorta and slower migration. Further inhibition of actin polymerization resulted in a greater decrease in sprouting angiogenesis, indicating that ECs employ two mechanisms for robust cell migration in vivo. Our study thus highlights an important role of hydrostatic pressure in tissue morphogenesis.

Vertebrate organs require locally adapted blood vessels^1,2. The gain of such organotypic vessel specializations is often deemed to be molecularly unrelated to the process of organ vascularization. Here, opposing this model, we reveal a molecular mechanism for brain-specific angiogenesis that operates under the control of Wnt7a/b ligands—well-known blood–brain barrier maturation signals^3,4,5. The control mechanism relies on Wnt7a/b-dependent expression of Mmp25, which we find is enriched in brain endothelial cells. CRISPR–Cas9 mutagenesis in zebrafish reveals that this poorly characterized glycosylphosphatidylinositol-anchored matrix metalloproteinase is selectively required in endothelial tip cells to enable their initial migration across the pial basement membrane lining the brain surface. Mechanistically, Mmp25 confers brain invasive competence by cleaving meningeal fibroblast-derived collagen IV α5/6 chains within a short non-collagenous region of the central helical part of the heterotrimer. After genetic interference with the pial basement membrane composition, the Wnt–β-catenin-dependent organotypic control of brain angiogenesis is lost, resulting in properly patterned, yet blood–brain-barrier-defective cerebrovasculatures. We reveal an organ-specific angiogenesis mechanism, shed light on tip cell mechanistic angiodiversity and thereby illustrate how organs, by imposing local constraints on angiogenic tip cells, can select vessels matching their distinctive physiological requirements.

Cell identities are defined by intrinsic transcriptional networks and spatio-temporal environmental factors. Here, we explored multiple factors that contribute to the identity of adipose stem cells, including anatomic location, microvascular neighborhood, and sex. Our data suggest that adipose stem cells serve a dual role as adipocyte precursors and fibroblast-like cells that shape the adipose tissue's extracellular matrix in an organotypic manner. We further find that adipose stem cells display sexual dimorphism regarding genes involved in estrogen signaling, homeobox transcription factor expression and the renin-angiotensin-aldosterone system. These differences could be attributed to sex hormone effects, developmental origin, or both. Finally, our data demonstrate that adipose stem cells are distinct from mural cells, and that the state of commitment to adipogenic differentiation is linked to their anatomic position in the microvascular niche. Our work supports the importance of sex and microvascular function in adipose tissue physiology. Several factors contribute to the identity of adipose stem cells, including anatomic location, microvascular neighborhood and sex. Here, authors find that adipose stem cells are sexual dimorphic cells with dual roles as preadipocytes and fibroblasts.

The dura, arachnoid and pia mater, as the constituent layers of the meninges, along with cerebrospinal fluid in the subarachnoid space and ventricles, are essential protectors of the brain and spinal cord. Complemented by immune cells, blood vessels, lymphatic vessels and nerves, these connective tissue layers have held many secrets that have only recently begun to be revealed. Each meningeal layer is now known to have molecularly distinct types of fibroblasts. Cerebrospinal fluid clearance through peripheral lymphatics and lymph nodes is well documented, but its routes and flow dynamics are debated. Advances made in meningeal immune functions are also debated. This Review considers the cellular and molecular structure and function of the dura, arachnoid and pia mater in the context of conventional views, recent progress, and what is uncertain or unknown. The hallmarks of meningeal pathophysiology are identified toward developing a more complete understanding of the meninges in health and disease. The authors review current knowledge of the molecular identity and functions of the dura, arachnoid and pial layers of meninges and controversial aspects of meningeal biology that deserve further study to resolve ongoing debates in the field.

For centuries, the meninges have been described as three membranes: the inner pia, middle arachnoid and outer dura. It was therefore sensational when in early 2023 Science magazine published a report of a previously unrecognized — 4th — meningeal membrane located between the pia and arachnoid. Multiple features were claimed for this new membrane: a single cell layer marked by the transcription factor Prox1 that formed a barrier to low molecular weight substances and separated the subarachnoid space (SAS) into two fluid-filled compartments, not one as previously described. These features were further claimed to facilitate unidirectional glymphatic cerebrospinal fluid transport. These claims were immediately questioned by several researchers as misinterpretations of the authors’ own data. The critics argued that (i) the 4th meningeal membrane as claimed did not exist as a separate structure but was part of the arachnoid, (ii) the “outer SAS” compartment was likely an artifactual subdural space created by the experimental procedures, and (iii) the 4th membrane barrier property was confused with the arachnoid barrier. Subsequent publications in late 2023 indeed showed that Prox1 + cells are embedded within the arachnoid and located immediately inside of and firmly attached to the arachnoid barrier cells by adherens junctions and gap junctions. In a follow-up study, published in this journal, the lead authors of the Science paper Kjeld Møllgård and Maiken Nedergaard reported additional observations they claim support the existence of a 4th meningeal membrane and the compartmentalization of the SAS into two non-communicating spaces. Their minor modification to the original paper was the 4th meningeal membrane was better observable at the ventral side of the brain than at the dorsal side where it was originally reported. The authors also claimed support for the existence of a 4th meningeal membrane in classical literature. Here, we outline multiple concerns over the new data and interpretation and argue against the claim there is prior support in the literature for a 4th meningeal membrane.

Lipoprotein lipase (LPL) and multiple regulators of LPL activity (e.g., APOC2 and ANGPTL4) are present in all vertebrates, but GPIHBP1—the endothelial cell (EC) protein that captures LPL within the subendothelial spaces and transports it to its site of action in the capillary lumen—is present in mammals but in not chickens or other lower vertebrates. In mammals, GPIHBP1 deficiency causes severe hypertriglyceridemia, but chickens maintain low triglyceride levels despite the absence of GPIHBP1. To understand intravascular lipolysis in lower vertebrates, we examined LPL expression in mouse and chicken hearts. In both species, LPL was abundant on capillaries, but the distribution of Lpl transcripts was strikingly different. In mouse hearts, Lpl transcripts were extremely abundant in cardiomyocytes but were barely detectable in capillary ECs. In chicken hearts, Lpl transcripts were absent in cardiomyocytes but abundant in capillary ECs. In zebrafish hearts, lpl transcripts were also in capillary ECs but not cardiomyocytes. In both mouse and chicken hearts, LPL was present, as judged by immunogold electron microscopy, in the glycocalyx of capillary ECs. Thus, mammals produce LPL in cardiomyocytes and rely on GPIHBP1 to transport the LPL into capillaries, whereas lower vertebrates produce LPL directly in capillary ECs, rendering an LPL transporter unnecessary.

While excessive production of reactive oxygen species (ROS) is a characteristic hallmark of numerous diseases, clinical approaches that ameliorate oxidative stress have been unsuccessful. Here, utilizing multi-omics, we demonstrate that in cardiomyocytes, mitochondrial isocitrate dehydrogenase (IDH2) constitutes a major antioxidative defense mechanism. Paradoxically reduced expression of IDH2 associated with ventricular eccentric hypertrophy is counterbalanced by an increase in the enzyme activity. We unveil redox-dependent sex dimorphism, and extensive mutual regulation of the antioxidative activities of IDH2 and NRF2 by a feedforward network that involves 2-oxoglutarate and L-2-hydroxyglutarate and mediated in part through unconventional hydroxy-methylation of cytosine residues present in introns. Consequently, conditional targeting of ROS in a murine model of heart failure improves cardiac function in sex- and phenotype-dependent manners. Together, these insights may explain why previous attempts to treat heart failure with antioxidants have been unsuccessful and open new approaches to personalizing and, thereby, improving such treatment.

The diverse etiologies of the genetic neurodegenerative disorder known as primary familial brain calcification have dimmed hopes for curative therapies. However, two new papers in Neuron provide a reason for optimism by identifying mechanisms involved in brain phosphate transport and a promising target for restoring phosphate balance in the brain.

Claudin-5 (CLDN5) is an endothelial tight junction protein essential for blood-brain barrier (BBB) formation. Abnormal CLDN5 expression is common in brain disease, and knockdown of Cldn5 at the BBB has been proposed to facilitate drug delivery to the brain. To study the consequences of CLDN5 loss in the mature brain, we induced mosaic endothelial-specific Cldn5 gene ablation in adult mice (Cldn5^iECKO). These mice displayed increased BBB permeability to tracers up to 10 kDa in size from 6 days post induction (dpi) and ensuing lethality from 10 dpi. Single-cell RNA sequencing at 11 dpi revealed profound transcriptomic differences in brain endothelial cells regardless of their Cldn5 status in mosaic mice, suggesting major non-cell-autonomous responses. Reactive microglia and astrocytes suggested rapid cellular responses to BBB leakage. Our study demonstrates a critical role for CLDN5 in the adult BBB and provides molecular insight into the consequences and risks associated with CLDN5 inhibition.

Cell isolation protocols from brain tissue include prolonged ex vivo processing durations, rendering them suboptimal for transcriptomic studies. Particularly for microglia and vascular cells, current isolation methods produce lower yields, necessitating addition of an enrichment step, and use of large tissue volumes - in most cases whole brain tissue - to obtain sufficient yields. Here, we developed a simple, rapid, and reproducible cell isolation method for generating singlecell suspensions from micro-dissected brain regions, enriched for microglia and vascular cells, without an enrichment step. Cells isolated using this method are suitable for molecular profiling studies using 10 x Genomics Chromium single-cell RNA sequencing with high reproducibility. Our method is valuable for longitudinal unbiased molecular profiling of microglia and vascular cells within different brain regions, spanning multiple time points across physiological development or disease progression.