| 1. |
|
|
| 2. |
- Bjornholm, Katrine Dahl, et al.
(författare)
-
A robust and efficient microvascular isolation method for multimodal characterization of the mouse brain vasculature
- 2023
-
Ingår i: CELL REPORTS METHODS. - : Elsevier. - 2667-2375. ; 3:3
-
Tidskriftsartikel (refereegranskat)abstract
- Studying disease-related changes in the brain vasculature is warranted due to its crucial role in supplying oxygen and nutrients and removing waste and due to the anticipated vascular dysfunction in brain dis-eases. To this end, we have developed a protocol for fast and simple isolation of brain vascular fragments without the use of transgenic reporters. We used it to isolate and analyze 22,515 cells by single-cell RNA sequencing. The cells distributed into 23 distinct clusters corresponding to all known vascular and perivas-cular cell types in the brain. Western blot analysis also suggested that the protocol is suitable for proteomic analysis. We further adapted it for the establishment of primary cell cultures. The protocol generated highly reproducible results. In conclusion, we have developed a simple and robust brain vascular isolation proto-col suitable for different experimental modalities, such as single-cell analyses, western blotting, and pri-mary cell culture.
|
|
| 3. |
- De La Fuente, Alerie Guzman, et al.
(författare)
-
Pericytes Stimulate Oligodendrocyte Progenitor Cell Differentiation during CNS Remyelination
- 2017
-
Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 20:8, s. 1755-1764
-
Tidskriftsartikel (refereegranskat)abstract
- The 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 Pdgfb(ret/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.
|
|
| 4. |
- Del Gaudio, Francesca, et al.
(författare)
-
Left ventricular hypertrophy and metabolic resetting in the Notch3-deficient adult mouse heart
- 2023
-
Ingår i: Scientific Reports. - : Springer Nature. - 2045-2322. ; 13:1
-
Tidskriftsartikel (refereegranskat)abstract
- The heart depends on a functional vasculature for oxygenation and transport of nutrients, and it is of interest to learn how primary impairment of the vasculature can indirectly affect cardiac function and heart morphology. Notch3-deficiency causes vascular smooth muscle cell (VSMC) loss in the vasculature but the consequences for the heart remain largely elusive. Here, we demonstrate that Notch3(-/-) mice have enlarged hearts with left ventricular hypertrophy and mild fibrosis. Cardiomyocytes were hypertrophic but not hyperproliferative, and the expression of several cardiomyocyte markers, including Tnt2, Myh6, Myh7 and Actn2, was altered. Furthermore, expression of genes regulating the metabolic status of the heart was affected: both Pdk4 and Cd36 were downregulated, indicating a metabolic switch from fatty acid oxidation to glucose consumption. Notch3(-/-) mice furthermore showed lower liver lipid content. Notch3 was expressed in heart VSMC and pericytes but not in cardiomyocytes, suggesting that a perturbation of Notch signalling in VSMC and pericytes indirectly impairs the cardiomyocytes. In keeping with this, Pdgfb(ret/ret) mice, characterized by reduced numbers of VSMC and pericytes, showed left ventricular and cardiomyocyte hypertrophy. In conclusion, we demonstrate that reduced Notch3 or PDGFB signalling in vascular mural cells leads to cardiomyocyte dysfunction.
|
|
| 5. |
- Greene, Chris, et al.
(författare)
-
Microvascular stabilization via blood-brain barrier regulation prevents seizure activity
- 2022
-
Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 13
-
Tidskriftsartikel (refereegranskat)abstract
- Blood-brain barrier (BBB) dysfunction is associated with worse epilepsy outcomes however the underlying molecular mechanisms of BBB dysfunction remain to be elucidated. Tight junction proteins are important regulators of BBB integrity and in particular, the tight junction protein claudin-5 is the most enriched in brain endothelial cells and regulates size-selectivity at the BBB. Additionally, disruption of claudin-5 expression has been implicated in numerous disorders including schizophrenia, depression and traumatic brain injury, yet its role in epilepsy has not been fully deciphered. Here we report that claudin-5 protein levels are significantly diminished in surgically resected brain tissue from patients with treatment-resistant epilepsy. Concomitantly, dynamic contrast-enhanced MRI in these patients showed widespread BBB disruption. We show that targeted disruption of claudin-5 in the hippocampus or genetic heterozygosity of claudin-5 in mice exacerbates kainic acid-induced seizures and BBB disruption. Additionally, inducible knockdown of claudin-5 in mice leads to spontaneous recurrent seizures, severe neuroinflammation, and mortality. Finally, we identify that RepSox, a regulator of claudin-5 expression, can prevent seizure activity in experimental epilepsy. Altogether, we propose that BBB stabilizing drugs could represent a new generation of agents to prevent seizure activity in epilepsy patients.
|
|
| 6. |
- He, Liqun, et al.
(författare)
-
Single-cell RNA sequencing of mouse brain and lung vascular and vessel-associated cell types
- 2018
-
Ingår i: Scientific Data. - : NATURE PUBLISHING GROUP. - 2052-4463. ; 5
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
|
|
| 7. |
- Henshall, Tanya L, et al.
(författare)
-
Notch3 Is Necessary for Blood Vessel Integrity in the Central Nervous System
- 2015
-
Ingår i: Arteriosclerosis, Thrombosis and Vascular Biology. - 1079-5642 .- 1524-4636. ; 35:2, s. 409-420
-
Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: Vascular smooth muscle cells (VSMC) are important for contraction, blood flow distribution, and regulation of blood vessel diameter, but to what extent they contribute to the integrity of blood vessels and blood-brain barrier function is less well understood. In this report, we explored the impact of the loss of VSMC in the Notch3(-/-) mouse on blood vessel integrity in the central nervous system.APPROACH AND RESULTS: Notch3(-/-) mice showed focal disruptions of the blood-brain barrier demonstrated by extravasation of tracers and accompanied by fibrin deposition in the retinal vasculature. This blood-brain barrier leakage was accompanied by a regionalized and patchy loss of VSMC, with VSMC gaps predominantly in arterial resistance vessels of larger caliber. The loss of VSMC appeared to be caused by progressive degeneration of VSMC resulting in a gradual loss of VSMC marker expression and a progressive acquisition of an aberrant VSMC phenotype closer to the gaps, followed by enhanced apoptosis and cellular disintegration in the gaps. Arterial VSMC were the only mural cell type that was morphologically affected, despite Notch3 being expressed also in pericytes. Transcriptome analysis of isolated brain microvessels revealed gene expression changes in Notch3(-/-) mice consistent with loss of arterial VSMC and presumably secondary transcriptional changes were observed in endothelial genes, which may explain the compromised vascular integrity.CONCLUSIONS: We demonstrate that Notch3 is important for survival of VSMC, and reveal a critical role for Notch3 and VSMC in blood vessel integrity and blood-brain barrier function in the mammalian vasculature.
|
|
| 8. |
- Holm, Tina, et al.
(författare)
-
Retro-inversion of certain cell-penetrating peptides causes severe cellular toxicity
- 2011
-
Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434 .- 0005-2736. ; 1808:6, s. 1544-1551
-
Tidskriftsartikel (refereegranskat)abstract
- Cell-penetrating peptides (CPPs) are a promising group of delivery vectors for various therapeutic agents but their application is often hampered by poor stability in the presence of serum. Different strategies to improve peptide stability have been exploited, one of them being "retro-inversion" (RI) of natural peptides. With this approach the stability of CPPs has been increased, thereby making them more efficient transporters. Several RI-CPPs were here assessed and compared to the corresponding parent peptides in different cell-lines. Surprisingly, treatment of cells with these peptides induced trypsin insensitivity and rapid severe toxicity in contrast to l-peptides. This was measured as reduced metabolic activity and condensed cell nuclei, in parity with the apoptosis inducing agent staurosporine. Furthermore, effects on mitochondrial network, focal adhesions, actin cytoskeleton and caspase-3 activation were analyzed and adverse effects were evident at 20μM peptide concentration within 4h while parent l-peptides had negligible effects. To our knowledge this is the first time RI peptides are reported to cause cellular toxicity, displayed by decreased metabolic activity, morphological changes and induction of apoptosis. Considering the wide range of research areas that involves the use of RI-peptides, this finding is of major importance and needs to be taken under consideration in applications of RI-peptides.
|
|
| 9. |
|
|
| 10. |
- Keller, Annika, et al.
(författare)
-
Mutations in the gene encoding PDGF-B cause brain calcifications in humans and mice
- 2013
-
Ingår i: Nature Genetics. - : Springer Science and Business Media LLC. - 1061-4036 .- 1546-1718. ; 45:9, s. 1077-
-
Tidskriftsartikel (refereegranskat)abstract
- Calcifications in the basal ganglia are a common incidental finding and are sometimes inherited as an autosomal dominant trait ( idiopathic basal ganglia calcification (IBGC)). Recently, mutations in the PDGFRB gene coding for the platelet-derived growth factor receptor beta (PDGF-R beta) were linked to IBGC. Here we identify six families of different ancestry with nonsense and missense mutations in the gene encoding PDGF-B, the main ligand for PDGF-R beta. We also show that mice carrying hypomorphic Pdgfb alleles develop brain calcifications that show age-related expansion. The occurrence of these calcium depositions depends on the loss of endothelial PDGF-B and correlates with the degree of pericyte and blood-brain barrier deficiency. Thus, our data present a clear link between Pdgfb mutations and brain calcifications in mice, as well as between PDGFB mutations and IBGC in humans.
|
|
| 11. |
- Kotini, Maria Paraskevi, et al.
(författare)
-
Sprouting and anastomosis in the Drosophila trachea and the vertebrate vasculature : Similarities and differences in cell behaviour
- 2019
-
Ingår i: Vascular pharmacology. - : Elsevier. - 1537-1891 .- 1879-3649. ; 112, s. 8-16
-
Tidskriftsartikel (refereegranskat)abstract
- Branching morphogenesis is a fascinating process whereby a simple network of biological tubes increases its complexity by adding new branches to existing ones, generating an enlarged structure of interconnected tubes. Branching morphogenesis has been studied extensively in animals and much has been learned about the regulation of branching at the cellular and molecular level. Here, we discuss studies of the Drosophila trachea and of the vertebrate vasculature, which have revealed how new branches are formed and connect (anastomose), leading to the establishment of complex tubular networks. We briefly describe the cell behaviour underlying tracheal and vascular branching. Although similar at many levels, the branching and anastomosis processes characterized thus far show a number of differences in cell behaviour, resulting in somewhat different tube architectures in these two organs. We describe the similarities and the differences and discuss them in the context of their possible developmental significance. We finish by highlighting some old and new data, which suggest that live imaging of the development of capillary beds in adult animals might reveal yet unexplored endothelial behaviour of endothelial cells.
|
|
| 12. |
- Lehto, Taavi, et al.
(författare)
-
A Peptide-based Vector for Efficient Gene Transfer In Vitro and In Vivo
- 2011
-
Ingår i: Molecular Therapy. - : Elsevier BV. - 1525-0016 .- 1525-0024. ; 19:8, s. 1457-1467
-
Tidskriftsartikel (refereegranskat)abstract
- Finding suitable nonviral delivery vehicles for nucleic acid-based therapeutics is a landmark goal in gene therapy. Cell-penetrating peptides (CPPs) are one class of delivery vectors that has been exploited for this purpose. However, since CPPs use endocytosis to enter cells, a large fraction of peptides remain trapped in endosomes. We have previously reported that stearylation of amphipathic CPPs, such as transportan 10 (TP10), dramatically increases transfection of oligonucleotides in vitro partially by promoting endosomal escape. Therefore, we aimed to evaluate whether stearyl-TP10 could be used for the delivery of plasmids as well. Our results demonstrate that stearyl-TP10 forms stable nanoparticles with plasmids that efficiently enter different cell-types in a ubiquitous manner, including primary cells, resulting in significantly higher gene expression levels than when using stearyl-Arg9 or unmodified CPPs. In fact, the transfection efficacy of stearyl-TP10 almost reached the levels of Lipofectamine 2000 (LF2000), however, without any of the observed lipofection-associated toxicities. Most importantly, stearyl-TP10/plasmid nanoparticles are nonimmunogenic, mediate efficient gene delivery in vivo, when administrated intramuscularly (i.m.) or intradermally (i.d.) without any associated toxicity in mice.
|
|
| 13. |
- Lund, Harald, et al.
(författare)
-
CD163+ macrophages monitor enhanced permeability at the blood-dorsal root ganglion barrier
- 2024
-
Ingår i: Journal of Experimental Medicine. - : Rockefeller University Press. - 0022-1007 .- 1540-9538. ; 221:2
-
Tidskriftsartikel (refereegranskat)abstract
- In dorsal root ganglia (DRG), macrophages reside close to sensory neurons and have largely been explored in the context of pain, nerve injury, and repair. However, we discovered that most DRG macrophages interact with and monitor the vasculature by sampling macromolecules from the blood. Characterization of the DRG vasculature revealed a specialized endothelial bed that transformed in molecular, structural, and permeability properties along the arteriovenous axis and was covered by macrophage-interacting pericytes and fibroblasts. Macrophage phagocytosis spatially aligned with peak endothelial permeability, a process regulated by enhanced caveolar transcytosis in endothelial cells. Profiling the DRG immune landscape revealed two subsets of perivascular macrophages with distinct transcriptome, turnover, and function. CD163(+) macrophages self-maintained locally, specifically participated in vasculature monitoring, displayed distinct responses during peripheral inflammation, and were conserved in mouse and man. Our work provides a molecular explanation for the permeability of the blood-DRG barrier and identifies an unappreciated role of macrophages as integral components of the DRG-neurovascular unit.
|
|
| 14. |
|
|
| 15. |
|
|
| 16. |
- Muhl, Lars, et al.
(författare)
-
The SARS-CoV-2 receptor ACE2 is expressed in mouse pericytes but not endothelial cells : Implications for COVID-19 vascular research
- 2022
-
Ingår i: Stem Cell Reports. - : Elsevier. - 2213-6711. ; 17:5, s. 1089-1104
-
Tidskriftsartikel (refereegranskat)abstract
- Humanized mouse models and mouse-adapted SARS-CoV-2 virus are increasingly used to study COVID-19 pathogenesis, so it is impor-tant to learn where the SARS-CoV-2 receptor ACE2 is expressed. Here we mapped ACE2 expression during mouse postnatal development and in adulthood. Pericytes in the CNS, heart, and pancreas express ACE2 strongly, as do perineurial and adrenal fibroblasts, whereas endothelial cells do not at any location analyzed. In a number of other organs, pericytes do not express ACE2, including in the lung where ACE2 instead is expressed in bronchial epithelium and alveolar type II cells. The onset of ACE2 expression is organ specific: in bronchial epithelium already at birth, in brain pericytes before, andin heart pericytes after postnatal day 10.5. Establishing the vascular localization of ACE2 expression is central to correctly interpret data from modeling COVID-19 in the mouse and may shed light on the cause of vascular COVID-19 complications.
|
|
| 17. |
- Munk, Anne Sofie, et al.
(författare)
-
PDGF-B Is Required for Development of the Glymphatic System
- 2019
-
Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; , s. 3-2969
-
Tidskriftsartikel (refereegranskat)abstract
- The glymphatic system is a highly polarized cerebrospinal fluid (CSF) transport system that facilitates the clearance of neurotoxic molecules through a brain-wide network of perivascular pathways. Herein we have mapped the development of the glymphatic system in mice. Perivascular CSF transport first emerges in hippocampus in newborn mice, and a mature glymphatic system is established in the cortex at 2 weeks of age. Formation of astrocytic endfeet and polarized expression of aquaporin 4 (AQP4) consistently coincided with the appearance of perivascular CSF transport. Deficiency of platelet-derived growth factor B (PDGF-B) function in the PDGF retention motif knockout mouse line Pdgfb ret/ret suppressed the development of the glymphatic system, whose functions remained suppressed in adulthood compared with wild-type mice. These experiments map the natural development of the glymphatic system in mice and define a critical role of PDGF-B in the development of perivascular CSF transport.
|
|
| 18. |
- Myrberg, Helena, et al.
(författare)
-
Design of a Tumor-Homing Cell-Penetrating Peptide
- 2008
-
Ingår i: Bioconjugate chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812. ; 19:1, s. 70-75
-
Tidskriftsartikel (refereegranskat)abstract
- Chemotherapy is often limited by toxicity to normal cells. Therefore, an ideal anticancer drug should discriminate between normal tissue and tumors. This would require a target receptor molecule mostly present in tumors. The cyclic peptide cCPGPEGAGC (PEGA) is a homing peptide that has previously been shown to accumulate in breast tumor tissue in mice. PEGA peptide does not cross the plasma membrane per se; however, when attached to the cell-penetrating peptide pVEC, the conjugate is taken up by different breast cancer cells in vitro. Additionally, the homing capacity of the PEGA-pVEC is conserved in vivo, where the conjugate mainly accumulates in blood vessels in breast tumor tissue and, consequently is taken up. Furthermore, we show that the efficacy of the anticancer drug, chlorambucil, is increased more than 4 times when the drug is conjugated to the PEGA-pVEC chimeric peptide. These data demonstrate that combining a homing sequence with a cell-penetrating sequence yields a peptide that combines the desirable properties of the parent peptides. Such peptides may be useful in diagnostics and delivery of therapeutic agents to an intracellular location in a specific tumor target tissue.
|
|
| 19. |
- Mäe, Maarja Andaloussi, et al.
(författare)
-
Prolonged systemic hyperglycemia does not cause pericyte loss and permeability at the mouse blood-brain barrier
- 2018
-
Ingår i: Scientific reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 8:1, s. 17462-
-
Tidskriftsartikel (refereegranskat)abstract
- Diabetes mellitus is associated with cognitive impairment and various central nervous system pathologies such as stroke, vascular dementia, or Alzheimer’s disease. The exact pathophysiology of these conditions is poorly understood. Recent reports suggest that hyperglycemia causes cerebral microcirculation pathology and blood-brain barrier (BBB) dysfunction and leakage. The majority of these reports, however, are based on methods including in vitro BBB modeling or streptozotocin-induced diabetes in rodents, opening questions regarding the translation of the in vitro findings to the in vivo situation, and possible direct effects of streptozotocin on the brain vasculature. Here we used a genetic mouse model of hyperglycemia (Ins2AKITA) to address whether prolonged systemic hyperglycemia induces BBB dysfunction and leakage. We applied a variety of methodologies to carefully evaluate BBB function and cellular integrity in vivo, including the quantification and visualization of specific tracers and evaluation of transcriptional and morphological changes in the BBB and its supporting cellular components. These experiments did neither reveal altered BBB permeability nor morphological changes of the brain vasculature in hyperglycemic mice. We conclude that prolonged hyperglycemia does not lead to BBB dysfunction, and thus the cognitive impairment observed in diabetes may have other causes.
|
|
| 20. |
- Mäe, Maarja Andaloussi, et al.
(författare)
-
Single-Cell Analysis of Blood-Brain Barrier Response to Pericyte Loss
- 2021
-
Ingår i: Circulation Research. - : Lippincott Williams & Wilkins. - 0009-7330 .- 1524-4571. ; 128:4, s. E46-E62
-
Tidskriftsartikel (refereegranskat)abstract
- Rationale: Pericytes are capillary mural cells playing a role in stabilizing newly formed blood vessels during development and tissue repair. Loss of pericytes has been described in several brain disorders, and genetically induced pericyte deficiency in the brain leads to increased macromolecular leakage across the blood-brain barrier (BBB). However, the molecular details of the endothelial response to pericyte deficiency remain elusive.Objective: To map the transcriptional changes in brain endothelial cells resulting from lack of pericyte contact at single-cell level and to correlate them with regional heterogeneities in BBB function and vascular phenotype.Methods and Results: We reveal transcriptional, morphological, and functional consequences of pericyte absence for brain endothelial cells using a combination of methodologies, including single-cell RNA sequencing, tracer analyses, and immunofluorescent detection of protein expression in pericyte-deficient adult Pdgfb(ret/ret) mice. We find that endothelial cells without pericyte contact retain a general BBB-specific gene expression profile, however, they acquire a venous-shifted molecular pattern and become transformed regarding the expression of numerous growth factors and regulatory proteins. Adult Pdgfb(ret/ret) brains display ongoing angiogenic sprouting without concomitant cell proliferation providing unique insights into the endothelial tip cell transcriptome. We also reveal heterogeneous modes of pericyte-deficient BBB impairment, where hotspot leakage sites display arteriolar-shifted identity and pinpoint putative BBB regulators. By testing the causal involvement of some of these using reverse genetics, we uncover a reinforcing role for angiopoietin 2 at the BBB.Conclusions: By elucidating the complexity of endothelial response to pericyte deficiency at cellular resolution, our study provides insight into the importance of brain pericytes for endothelial arterio-venous zonation, angiogenic quiescence, and a limited set of BBB functions. The BBB-reinforcing role of ANGPT2 (angiopoietin 2) is paradoxical given its wider role as TIE2 (TEK receptor tyrosine kinase) receptor antagonist and may suggest a unique and context-dependent function of ANGPT2 in the brain.
|
|
| 21. |
- Mäe, Maarja, et al.
(författare)
-
A stearylated CPP for delivery of splice correcting oligonucleotides using a non-covalent co-incubation strategy
- 2009
-
Ingår i: Journal of Controlled Release. - : Elsevier BV. - 0168-3659 .- 1873-4995. ; 134:3, s. 221-227
-
Tidskriftsartikel (refereegranskat)abstract
- Aberrations in splicing patterns play a significant role in several diseases, and splice correction, together with other forms of gene regulation, is consequently an emerging therapeutic target. In order to achieve successful oligonucleotide transfection, efficient delivery vectors are generally necessary. In this study we present one such vector, the chemically modified cell-penetrating peptide (CPP) TP10, for efficient delivery of a splice-correcting 2'-OMe RNA oligonucleotide. Utilizing a functional splice correction assay, we assessed the transfection efficiency of non-covalent complexes of oligonucleotides and stearylated or cysteamidated CPPs. Stearylation of the CPPs Arg9 and penetratin, as well as cysteamidation of MPG and TP10, did not improve transfection, whereas the presence of an N-terminal stearyl group on TP10 improved delivery efficiency remarkably compared to the unmodified peptide. The splice correction levels observed with stearyl-TP10 are in fact in parity with the effects seen with the commercially available transfection agent Lipofectamine (TM) 2000. However, the inherent toxicity associated with cationic lipid-based transfections can be completely eliminated when using the stearylated TP10, making this vector highly promising for non-covalent delivery of negatively charged oligonucleotides.
|
|
| 22. |
- Mäe, Maarja, et al.
(författare)
-
Chemically modified cell-penetrating peptides for the delivery of nucleic acids
- 2009
-
Ingår i: Expert Opinion on Drug Delivery. - : Informa Healthcare. - 1742-5247 .- 1744-7593. ; 6:11, s. 1195-1205
-
Tidskriftsartikel (refereegranskat)abstract
- Short nucleic acids targeting biologically important RNAs and plasmids have been shown to be promising future therapeutics; however, their hydrophilic nature greatly limits their utility in clinics and therefore efficient delivery vectors are greatly needed. Cell-penetrating peptides (CPPs) are relatively short amphipathic and/or cationic peptides that are able to transport various biologically active molecules inside mammalian cells, both in vitro and in vivo, in a seemingly non-toxic fashion. Although CPPs have proved to be appealing drug delivery vehicles, their major limitation in nucleic acid delivery is that most of the internalized peptide-cargo is entrapped in endosomal compartments following endocytosis and the bioavailability is therefore severely reduced. Several groups are working towards overcoming this obstacle and this review highlights the evidence that by introducing chemical modification in CPPs, the bioavailability of delivered nucleic acids increases significantly.
|
|
| 23. |
- Mäe, Maarja, et al.
(författare)
-
Design of a tumor homing cell-penetrating peptide for drug delivery
- 2009
-
Ingår i: International Journal of Peptide Research and Therapeutics. - : Springer Science and Business Media LLC. - 1573-3149 .- 1573-3904. ; 15:1, s. 11-15
-
Tidskriftsartikel (refereegranskat)abstract
- The major drawbacks with conventional cancer chemotherapy are the lack of satisfactory specificity towards tumor cells and poor antitumor activity. In order to improve these characteristics, chemotherapeutic drugs can be conjugated to targeting moieties e.g. to peptides with the ability to recognize cancer cells. We have previously reported that combining a tumor homing peptide with a cell-penetrating peptide yields a chimeric peptide with tumor cell specificity that can carry cargo molecules inside the cells. In the present study, we have used a linear breast tumor homing peptide, CREKA, in conjunction with a cell-penetrating peptide, pVEC. This new chimeric peptide, CREKA–pVEC, is more convenient to synthesize and moreover it is better in translocating cargo molecules inside cancer cells as compared to previously published PEGA–pVEC peptide. This study demonstrates that CREKA–pVEC is a suitable vehicle for targeted intracellular delivery of a DNA alkylating agent, chlorambucil, as the chlorambucil–peptide conjugate was substantially better at killing cancer cells in vitro than the anticancer drug alone.
|
|
| 24. |
|
|
| 25. |
- Mäe, Maarja, et al.
(författare)
-
Internalisation of cell-penetrating peptides into tobacco protoplasts
- 2005
-
Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434 .- 0005-2736. ; 1669:2, s. 101-107
-
Tidskriftsartikel (refereegranskat)abstract
- Cells are protected from the surrounding environment by plasma membrane which is impenetrable for most hydrophilic molecules. In the last 10 years cell-penetrating peptides (CPPs) have been discovered and developed. CPPs enter mammalian cells and carry cargo molecules over the plasma membrane with a molecular weight several times their own. Known transformation methods for plant cells have relatively low efficiency and require improvement. The possibility to use CPPs as potential delivery vectors for internalisation in plant cells has been studied in the present work. We analyse and compare the uptake of the fluorescein-labeled CPPs, transportan, TP10, penetratin and pVEC in Bowes human melanoma cells and Nicotiana tabacum cultivar (cv.) SR-1 protoplasts (plant cells without cell wall). We study the internalisation efficiency of CPPs with fluorescence microscopy, spectrofluorometry and fluorescence-activated cell sorter (FACS). All methods indicate, for the first time, that these CPPs can internalise into N. tabacum cv. SR-1 protoplasts. Transportan has the highest uptake efficacy among the studied peptides, both in mammalian cells and plant protoplast. The internalisation of CPPs by plant protoplasts may open up a new effective method for transfection in plants.
|
|
| 26. |
- Mäe, Maarja, 1979-
(författare)
-
Rational modifications of cell-penetrating peptides for drug delivery : Applications in tumor targeting and oligonucleotide delivery
- 2009
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- High molecular weight biomolecules are becoming important in the development of new therapeutics. However, their size and nature creates a major limitation for their application – poor penetration through biological membranes. A new class of peptides, cell-penetrating peptides (CPPs), has shown the capability to transport various macromolecules inside the cells. However, there are at least two limiting factors for successful application of CPPs: the lack of cell-type specificity and restricted bioavailability resulting from endocytic uptake of CPPs and entrapment in endosomal compartments. This thesis aims at designing delivery vehicles for therapeutic substances. In papers I-III, the CPPs have been rationally modified in order to achieve in vivo selectivity towards cancer cells. The first two papers employ tumor homing peptides as targeting moieties coupled to the N-termini of CPPs. In the third paper, a CPP is C-terminally prolonged with a matrix metalloproteinase 2 (MMP-2) specific cleavage site followed by an inactivating amino acid sequence. In tissues overexpressing MMP-2, i. e. in proximity to cancer, the CPP is activated after proteolytic removal of the inactivating sequence, thus the cargo can be transported inside the cells. In paper IV, several CPPs have been N-terminally modified with a stearyl moiety and applied for the delivery of splice-correcting oligonucleotides. We show that stearyl-TP10 is as effective in oligonucleotide delivery as Lipofectamine™ 2000. Moreover, stearyl-TP10 has preserved efficacy in serum and is not toxic to cells. In conclusion, the rational modifications of CPPs greatly potentiate their application in cargo delivery both in vitro and in vivo.
|
|
| 27. |
|
|
| 28. |
- Nahar, Khayrun, et al.
(författare)
-
Astrocyte-microglial association and matrix composition are common events in the natural history of primary familial brain calcification
- 2020
-
Ingår i: Brain Pathology. - : Wiley. - 1015-6305 .- 1750-3639. ; 30:3, s. 446-64
-
Tidskriftsartikel (refereegranskat)abstract
- Primary familial brain calcification (PFBC) is an age-dependent and rare neurodegenerative disorder characterized by microvascular calcium phosphate deposits in the deep brain regions. Known genetic causes of PFBC include loss-of-function mutations in genes involved in either of three processes-platelet-derived growth factor (PDGF) signaling, phosphate homeostasis or protein glycosylation-with unclear molecular links. To provide insight into the pathogenesis of PFBC, we analyzed murine models of PFBC for the first two of these processes in Pdgfb(ret/ret) and Slc20a2(-/-) mice with regard to the structure, molecular composition, development and distribution of perivascular calcified nodules. Analyses by transmission electron microscopy and immunofluorescence revealed that calcified nodules in both of these models have a multilayered ultrastructure and occur in direct contact with reactive astrocytes and microglia. However, whereas nodules in Pdgfb(ret/ret) mice were large, solitary and smooth surfaced, the nodules in Slc20a2(-/-) mice were multi-lobulated and occurred in clusters. The regional distribution of nodules also differed between the two models. Proteomic analysis and immunofluorescence stainings revealed a common molecular composition of the nodules in the two models, involving proteins implicated in bone homeostasis, but also proteins not previously linked to tissue mineralization. While the brain vasculature of Pdgfb(ret/ret) mice has been reported to display reduced pericyte coverage and abnormal permeability, we found that Slc20a2(-/-) mice have a normal pericyte coverage and no overtly increased permeability. Thus, lack of pericytes and increase in permeability of the blood-brain barrier are likely not the causal triggers for PFBC pathogenesis. Instead, gene expression and spatial correlations suggest that astrocytes are intimately linked to the calcification process in PFBC.
|
|
| 29. |
|
|
| 30. |
- Pietilä, Riikka, et al.
(författare)
-
Molecular anatomy of adult mouse leptomeninges
- 2023
-
Ingår i: Neuron. - : Elsevier. - 0896-6273 .- 1097-4199. ; 111:23
-
Tidskriftsartikel (refereegranskat)abstract
- Leptomeninges, consisting of the pia mater and arachnoid, form a connective tissue investment and barrier enclosure of the brain. The exact nature of leptomeningeal cells has long been debated. In this study, we iden-tify five molecularly distinct fibroblast-like transcriptomes in cerebral leptomeninges; link them to anatomically distinct cell types of the pia, inner arachnoid, outer arachnoid barrier, and dural border layer; and contrast them to a sixth fibroblast-like transcriptome present in the choroid plexus and median eminence. Newly identified transcriptional markers enabled molecular characterization of cell types responsible for adherence of arach-noid layers to one another and for the arachnoid barrier. These markers also proved useful in identifying the molecular features of leptomeningeal development, injury, and repair that were preserved or changed after traumatic brain injury. Together, the findings highlight the value of identifying fibroblast transcriptional subsets and their cellular locations toward advancing the understanding of leptomeningeal physiology and pathology.
|
|
| 31. |
- Vanlandewijck, Michael, et al.
(författare)
-
A molecular atlas of cell types and zonation in the brain vasculature
- 2018
-
Ingår i: Nature. - : NATURE PUBLISHING GROUP. - 0028-0836 .- 1476-4687. ; 554:7693, s. 475-480
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
|
|
| 32. |
- Vazquez-Liebanas, Elisa, et al.
(författare)
-
Adult-induced genetic ablation distinguishes PDGFB roles in blood-brain barrier maintenance and development
- 2022
-
Ingår i: Journal of Cerebral Blood Flow and Metabolism. - : SAGE Publications. - 0271-678X .- 1559-7016. ; 42:2, s. 264-279
-
Tidskriftsartikel (refereegranskat)abstract
- Platelet-derived growth factor B (PDGFB) released from endothelial cells is indispensable for pericyte recruitment during angiogenesis in embryonic and postnatal organ growth. Constitutive genetic loss-of-function of PDGFB leads to pericyte hypoplasia and the formation of a sparse, dilated and venous-shifted brain microvasculature with dysfunctional blood-brain barrier (BBB) in mice, as well as the formation of microvascular calcification in both mice and humans. Endothelial PDGFB is also expressed in the adult quiescent microvasculature, but here its importance is unknown. We show that deletion of Pdgfb in endothelial cells in 2-months-old mice causes a slowly progressing pericyte loss leading, at 12–18 months of age, to ≈50% decrease in endothelial:pericyte cell ratio, ≈60% decrease in pericyte longitudinal capillary coverage and >70% decrease in pericyte marker expression. Similar to constitutive loss of Pdgfb, this correlates with increased BBB permeability. However, in contrast to the constitutive loss of Pdgfb, adult-induced loss does not lead to vessel dilation, impaired arterio-venous zonation or the formation of microvascular calcifications. We conclude that PDFGB expression in quiescent adult microvascular brain endothelium is critical for the maintenance of pericyte coverage and normal BBB function, but that microvessel dilation, rarefaction, arterio-venous skewing and calcification reflect developmental roles of PDGFB.
|
|
| 33. |
- Vázquez-Liébanas, Elisa, et al.
(författare)
-
Mosaic deletion of claudin-5 reveals rapid non-cell-autonomous consequences of blood-brain barrier leakage
- 2024
-
Ingår i: Cell Reports. - : Elsevier. - 2211-1247. ; 43:3
-
Tidskriftsartikel (refereegranskat)abstract
- 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 (Cldn5iECKO). 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.
|
|
| 34. |
- Wang, Yixin, et al.
(författare)
-
Smooth muscle cell recruitment to lymphatic vessels requires PDGFB and impacts vessel size but not identity
- 2017
-
Ingår i: Development. - : COMPANY OF BIOLOGISTS LTD. - 0950-1991 .- 1477-9129. ; 144:19, s. 3590-3601
-
Tidskriftsartikel (refereegranskat)abstract
- Tissue fluid drains through blind-ended lymphatic capillaries, via smooth muscle cell (SMC)-covered collecting vessels into venous circulation. Both defective SMC recruitment to collecting vessels and ectopic recruitment to lymphatic capillaries are thought to contribute to vessel failure, leading to lymphedema. However, mechanisms controlling lymphatic SMC recruitment and its role in vessel maturation are unknown. Here, we demonstrate that platelet-derived growth factor B (PDGFB) regulates lymphatic SMC recruitment in multiple vascular beds. PDGFB is selectively expressed by lymphatic endothelial cells (LECs) of collecting vessels. LEC-specific deletion of Pdgfb prevented SMC recruitment causing dilation and failure of pulsatile contraction of collecting vessels. However, vessel remodelling and identity were unaffected. Unexpectedly, Pdgfb overexpression in LECs did not induce SMC recruitment to capillaries. This was explained by the demonstrated requirement of PDGFB extracellular matrix (ECM) retention for lymphatic SMC recruitment, and the low presence of PDGFB-binding ECM components around lymphatic capillaries. These results demonstrate the requirement of LEC-autonomous PDGFB expression and retention for SMC recruitment to lymphatic vessels, and suggest an ECM-controlled checkpoint that prevents SMC investment of capillaries, which is a common feature in lymphedematous skin.
|
|
