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3.8.4: Extracellular Matrix of Animal Cells - Biology

3.8.4: Extracellular Matrix of Animal Cells - Biology


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The extracellular matrix of animal cells holds cells together to form a tissue and allow tissues to communicate with each other.

LEARNING OBJECTIVES

Explain the role of the extracellular matrix in animal cells

Key Points

  • The extracellular matrix of animal cells is made up of proteins and carbohydrates.
  • Cell communication within tissue and tissue formation are main functions of the extracellular matrix of animal cells.
  • Tissue communication is kick-started when a molecule within the matrix binds a receptor; the end results are conformational changes that induce chemical signals that ultimately change activities within the cell.

Key Terms

  • collagen: Any of more than 28 types of glycoprotein that forms elongated fibers, usually found in the extracellular matrix of connective tissue.
  • proteoglycan: Any of many glycoproteins that have heteropolysaccharide side chains
  • extracellular matrix: All the connective tissues and fibres that are not part of a cell, but rather provide support.

Extracellular Matrix of Animal Cells

Most animal cells release materials into the extracellular space. The primary components of these materials are proteins. Collagen is the most abundant of the proteins. Its fibers are interwoven with carbohydrate-containing protein molecules called proteoglycans. Collectively, these materials are called the extracellular matrix. Not only does the extracellular matrix hold the cells together to form a tissue, but it also allows the cells within the tissue to communicate with each other.

How does this cell communication occur? Cells have protein receptors on the extracellular surfaces of their plasma membranes. When a molecule within the matrix binds to the receptor, it changes the molecular structure of the receptor. The receptor, in turn, changes the conformation of the microfilaments positioned just inside the plasma membrane. These conformational changes induce chemical signals inside the cell that reach the nucleus and turn “on” or “off” the transcription of specific sections of DNA. This affects the production of associated proteins, thus changing the activities within the cell.

An example of the role of the extracellular matrix in cell communication can be seen in blood clotting. When the cells lining a blood vessel are damaged, they display a protein receptor called tissue factor. When a tissue factor binds with another factor in the extracellular matrix, it causes platelets to adhere to the wall of the damaged blood vessel and stimulates the adjacent smooth muscle cells in the blood vessel to contract (thus constricting the blood vessel). Subsequently, a series of steps are initiated which then prompt the platelets to produce clotting factors.


Extracellular matrix structure ☆

Extracellular matrix (ECM) is a non-cellular three-dimensional macromolecular network composed of collagens, proteoglycans/glycosaminoglycans, elastin, fibronectin, laminins, and several other glycoproteins. Matrix components bind each other as well as cell adhesion receptors forming a complex network into which cells reside in all tissues and organs. Cell surface receptors transduce signals into cells from ECM, which regulate diverse cellular functions, such as survival, growth, migration, and differentiation, and are vital for maintaining normal homeostasis. ECM is a highly dynamic structural network that continuously undergoes remodeling mediated by several matrix-degrading enzymes during normal and pathological conditions. Deregulation of ECM composition and structure is associated with the development and progression of several pathologic conditions. This article emphasizes in the complex ECM structure as to provide a better understanding of its dynamic structural and functional multipotency. Where relevant, the implication of the various families of ECM macromolecules in health and disease is also presented.


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Introduction

In mammals, the antral ovarian follicle is filled with follicular fluid (FF), which provides an optimal environment for oocyte growth, meiotic maturation, and the acquisition of oocyte competence for embryonic development (1, 2). The FF is derived from both blood plasma and the secretions of different types of somatic follicular cells (cumulus, granulosa, and thecal layers). The active exchange of molecular factors occurs between follicular cells and the oocyte via different mechanisms of cell-to-cell communication including extracellular vesicles (EVs) (3𠄶). EVs are enclosed within membranes and can be classified into three principal groups according to their biogenesis and size, as follows: exosomes (30� nm), macrovesicles (100𠄱,000 nm), and apoptotic bodies (7). Exosomes are released to the extracellular space from intracellular multivesicular bodies on fusion with the plasma membrane. They contain different molecular cargo, which includes proteins, lipids, and different types of RNAs and DNA. These factors can be transferred to target cells to facilitate cell-to-cell communication and signal transmission in different physiological systems, including reproductive organs (4, 7, 8).

Recently, EVs have been identified in most types of maternal reproductive fluids (follicular, oviductal, and uterine fluids) (4, 9�). It has been suggested that exosomes, like extracellular vesicles identified in the follicular fluid (ffEVs), mediate molecular signaling between follicle cells and may modulate the functions of target cells [reviewed by Tesfaye et al. (6)]. It has been shown that bovine ffEVs are capable of stimulating granulosa cell proliferation (14) and cumulus expansion in vitro (15). These effects were associated with the sizes of follicles from which ffEVs originated and revealed that ffEVs from small follicles (3𠄶 mm) were preferentially taken up by follicular cells vs. larger follicles (14). Further, research revealed that these follicles affected oocyte competence to a greater degree than large follicles (16), and displayed increased blastocyst rates (16) and modulated gene expression in cumulus cells (CC) (17). However, the addition of ffEVs during in vitro maturation (IVM) did not affect oocyte maturation rates (17). In contrast, when ffEV supplementation was assessed throughout the IVM of heat-stressed oocytes, increases in the rates of cell cleavage and embryo development were observed (18). Interestingly, rates were similar to those associated FF supplementation, which improved the quality of blastocysts produced in vitro (19). Taken together, recent studies have shown that ffEVs play important roles in the process of oocyte maturation by affecting functional mechanisms of meiotic division, cytoplasm maturation, and stress protection via different pathways. Moreover, molecules that are released from surrounding follicular cells into FF via ffEVs could serve as non-invasive molecular markers of oocyte competence (6).

To date, most studies of the molecular cargo of ffEVs have focused on ffEV miRNA and their roles in the regulation of target cell gene expression after EV uptake by granulosa cells (GG) and cumulus-oocyte complex (COC) (11, 17, 20�). A number of miRNAs have been determined to be up- or downregulated in small subordinate follicle ffEVs compared with larger ones. In addition, some miRNAs of ffEV have been associated with cellular proliferation and inflammatory response pathways (23). Differences have been also observed between ffEV miRNAs of hyperstimulated and unstimulated heifers. Differentially expressed genes identified were associated with genes involved in oocyte meiosis and MAPK and TGF-beta signaling pathways (22). Furthermore, different cargo of ffEV miRNAs have been identified in follicles with low and high progesterone levels, and some activate numerous signaling pathways in CC or COCs after 9 h IVM (17). In post-calving cows, miRNA expression patterns of ffEVs of cows with different degrees of energy balance differed. Further, differential expression of miRNA-targeting genes involved in TGF-beta, mTOR, and PI3K signaling, apoptosis, the cell cycle, adherent junction, and other biological pathways important for reproductive functions was observed (24).

Data regarding the molecular components of EVs and their release from different cell types that were determined in vivo, in vitro, and from the assessment of variety of biological fluids from different species have been deposited in the public online database, Vesiclepedia (25). In 2019, this database contained 10,500 miRNA entries, 27,600 mRNA entries, and 350,000 protein entries (26). Compared with other species, proteomic data derived from bovine EVs are scarce. In fact, only 1,562 protein IDs from bovine EVs have been reported up to date, compared with 12,800 protein IDs that have been identified from human EVs. In cows, EV proteins have previously been identified from plasma (27), urine, saliva, milk (28), and oviduct fluid (10). However, no comprehensive analysis of the bovine ffEV protein cargo has yet been performed. In contrast, the protein cargo of ffEVs has been assessed in equine and human ffEVs, and 73 and 662 proteins were identified, respectively (20, 29). Moreover, a comparison of the abundance of ffEV proteins in control and polycystic ovary syndrome ovaries could potentially identify potential markers of oocyte quality (29).

FfEVs and their cargo have the potential to affect oocyte competence acquisition, which is necessary for ensuring optimal embryo development, and they are potential markers of oocyte quality. Despite its potential utility, minimal data regarding protein cargo of bovine ffEVs are available. Therefore, the present study aimed to characterize the protein cargo of bovine ffEVs, particularly within exosomes. Moreover, because follicles contain different types of follicular cells (granulosa, theca, and CC), and because enclosed oocytes are within FF, we aimed to reveal the origin of ffEVs by analyzing ffEV proteins and corresponding mRNA expression levels in follicular cells.


3.8.4: Extracellular Matrix of Animal Cells - Biology

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- 많은 세포 조직을 지지해주는세포외 기질또는 ECN는상호 연결된 네트워크로서섬유와 바탕 물질로 구성되어 있으며이의 대부분은 간질 유체로서세포 사이 공간을 메꾸는데, 이는 결합 조직 섬유와모세 혈관입니다각 미세 환경의 분자 구성은국부 세포에 의해 분비되며섬유아 세포에서 대식 세포에 이르며차이가 정확한 구성과물리적 특성에 나타나게 됩니다예를 들어, 기질을 채우고 있는 것은 중합된글리코아미노글리칸또는 GAG 입니다이들이 물과 다른 단백질을 결합하고프로테오글리칸이 형성됩니다가장 일반적인 콘드로이틴 황산염은연골과 뼈에 필수적이며점진적인 손실은 무릎 관절염을 유발합니다엘라스틴이라는 또 다른 단백질은 신축성에 기여하여근육과 피부가 유연해집니다반면, 단단함의 제공은 길고 두꺼운 섬유로 된당단백으로 콜라겐이라고 합니다매우 흔하지만, 특히 이를 중요시하는 대상은힘줄과 같이 강하고 매우 거친 조직으로근육과 뼈를 연결합니다접착 역할은 파이브로넥틴인 세포 접착 단백질이며세포를 달라붙게 하여다른 기질 요소에 부착되는데, 콜라겐과GAG 및 인테그린이 포합됩니다막 단백질은 세포를 그 환경에 연결시켜주며신호전달 캐스케이드에서 중요한 역할을 합니다궁극적으로, 어디에 위치해 있더라도ECN은 서포트 네트워크를 아우르며이는 세포와 조직 주변의 통합에 필요한 것입니다

4.13: 세포외 기질

생체조직(tissue)의 조직(organization)을 유지하기 위해 여러 동물 세포는 세포외 기질(extracellular matrix, 줄여서 ECM)을 구성하는 구조적인 분자에 둘러싸여 있습니다. ECM에 있는 분자는 조직의 구조적 온전함을 유지하고 특정 조직의 놀라운 특성도 유지합니다.

세포외 기질의 구성

세포외 기질(이하 ECM)은 일반적으로 접지 물질(ground substance), 젤과 같은 액체, 섬유 성분, 그리고 여러 구조적, 기능적으로 다양한 분자로 구성되는데, 여기서 이 분자는 글리코사미노글리칸(glycosaminoglycan, 줄여서 GAG. 이하 GAG라 칭함)이라 불리는 다당류(polysaccharide)를 포함합니다. GAG는 세포외 기질의 대부분을 차지하고 종종 질량에 비해 큰 부피를 차지하는데 이로 인해 엄청난 압축력을 견딜 수 있는 기질(matrix)이 발생합니다. 대부분의 GAG는 단백질에 결합해 프로테오글리칸(proteoglycans)을 만드는데 해당분자는 분자 자신의 양전하에 따라 나트륨 이온을 간직해 물을 끌어들이게 되어 ECM에 수분을 공급합니다.

ECM은 또한 ECM의 기본 단백질 구성요소인 콜라겐(collagen) 같이 단단한 섬유를 포함합니다. 콜라겐은 동물에게 있는 가장 풍부한 단백질로 질량 기준 단백질의 25%를 차지합니다. 구조적으로 유사한 다양한 콜라겐은 여러 조직에 인장 강도(tensile strength)를 부여합니다.

특히, 피부, 혈관, 폐와 같은 조직은 생리적인 역할을 수행하기 위해 강하고 신축성이 있어야 합니다. 엘라스틴(elastin)이라고 불리는 단백질은 특정 섬유질에 늘어지고 수축하는 능력을 부여합니다. 파이브로넥틴(fibronectin 피브로넥틴)은 세포막을 포괄하는 단백질 (특히 인테그린(integrin))에 직접 결합해 ECM과 세포막을 연결하기 때문에 세포부착(cell adhesion)에 중요한 당단백질(glycoprotein)입니다. 인테그린은 또한 세포 내 반응을 끌어낼 수 있는 콜라겐과 상호작용합니다.

세포외 기질 구성은 조직과 세포 유형에 따라 다릅니다

ECM 내부 분자의 구성과 상대적 비율은 세포가 있는 조직(tissue)의 위치, 생리학적 기능, 인접 세포 유형에 따라 결정됩니다. ECM의 이러한 특정 분자 구성은 국소적 미세환경(microenvironment)이라고 합니다. 특정 조직의 세포는 주변 ECM을 결정하는 분자를 분비합니다. 예를 들어, 장 세포는 자신을 둘러싸고 있는 기질에 필요한 분자를 합성, 수정, 분비하는 반면, 골 세포는 인간 뼈에 단단함을 부여하는 ECM 분자를 생성합니다. 이렇게 여러 조직 내 다양한 ECM 구성은 조직의 다양한 역할과 기능에 따른 특성을 생성합니다.

세포외 기질은 세포 통신에 관여 할 수 있습니다

세포와 국소 ECM 간의 상호작용이 세포 내 영향을 미치는 것은 알려져 있습니다. 예를 들어, 막의 안팎에 있는 인테그린 분자에 작용하는 힘은 세포 내 액토미오신(actomyosin) 네트워크의 활성화를 초래할 수 있습니다. 이런 현상은 세포 이동, 분열 및 기타 세포 반응을 촉진할 수 있습니다. 이러한 세포반응 중 일부는 유전자 발현(gene expression)과 세포 신호전달체계의 변화를 포함합니다. 마찬가지로 인테그린은 세포 내 정보를 세포 외부로 전달할 수 있습니다. 또한 ECM은 ECM 분해 시 방출될 수 있는 신호 분자에 결합하는 것으로 알려져 있습니다.

세포외 기질의 재구성

동물 세포는 ECM을 분해하고 재구성할 수 있는 능력이 있어야 합니다. 특히 조직 수복(tissue repair)과 성장의 경우 이에 해당합니다. 따라서 세포는 일반적으로 ECM을 분해하는 데 필요한 효소를 가지고 있습니다. 이런 효소는 콜라겐과 파이브로넥틴과 같은 단백질을 분해하기 위해 다른 효소와 함께 작용하는 기질 금속단백질분해효소(matrix metalloproteases, 줄여서 MMP)를 포함합니다. ECM 분해 및 재구성은 혈관 생성을 포함, 건강한 조직 성장에 중요합니다. 단점은 ECM 재구성은 암세포가 온몸으로 전이하는데 이바지하는 점입니다.

Frantz, Christian, Kathleen M. Stewart, and Valerie M. Weaver. &ldquoThe Extracellular Matrix at a Glance.&rdquo Journal of Cell Science 123, no. 24 (December 15, 2010): 4195&ndash4200. [Source]

Alberts, Bruce, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter. &ldquoThe Extracellular Matrix of Animals.&rdquo Molecular Biology of the Cell. 4th Edition, 2002. [Source]


DISCUSSION

Overview

We have conducted a systematic large-scale data synthesis to quantitatively characterize the amounts, kinetics and mechanisms of MSAR under normal and pathological conditions. From 228 systematically selected studies, we extracted 123 estimates of absolute and 212 estimates of relative amount of ATP released, 74 kinetic time-series, 592 pharmacological and 89 genetic intervention outcomes, and 51 pathophysiological comparisons. Using a meta-analytic approach, we have established that mechanically stimulated mammalian cells release 38.6 (95% CI: 18.2–81.8) amol ATP/cell, with a characteristic time constant of 32 s (95% CI: 16–66) measured using real-time recording methods. We have found that MSAR is a universally conserved phenomenon in mammalian cells, and that cells from different species, embryonic origin and most organ systems release similar amounts of ATP when mechanically stimulated. Our data-driven summary of MSAR mechanisms allowed us to infer tissue-level generalizations that suggest the existence of common and tissue-specific routes of ATP release, and to identify conserved cell type-independent signaling patterns. We have found that inflammation and injury were associated with increased MSAR, whereas hereditary and metabolic conditions resulted in attenuated ATP release. Importantly, several lines of evidence including (1) differences in release kinetics, (2) implicated mechanisms and (3) pathophysiological effects in PKD, suggest that cells can discriminate between stretch- and shear-related forces. Thus, consolidating and quantifying over 25 years of basic research data generated in 64 unique cell types derived from 12 organ systems and stimulated by 9 distinct force applications allowed us to generate novel testable hypotheses, and provide evidence-driven recommendations for translational studies.

Study limitations

The studies included in this meta-analysis were highly heterogeneous however, this was expected due to the higher methodological variability in exploratory basic science studies (Bradbury and Plückthun, 2015 Fontoura-Andrade et al., 2017 Soehnlein and Silvestre-Roig, 2017). Importantly, accounting for interstudy differences in ATP calibration and recording methods allowed us to dramatically reduce heterogeneity. We found minimal evidence of publication bias. We also demonstrated that the quality of the studies did not significantly affect study-level outcomes, despite quality scores varying substantially across studies. It is known that subgroup analyses come at the expense of lower statistical power however, the large number of available datasets permitted statistically powered analysis for numerous secondary outcomes (Jackson and Turner, 2017). Analysis of MSAR mechanisms was limited by overlapping and off-target effects of many inhibitors, as well as lack of within-study inhibitor validation and over-reliance on assumed pharmacological targets. We minimized false-positive outcomes by applying random effects meta-analytic models and considering Bonferroni adjustments for multiple comparison analyses (Glass, 1986). There remains a distinct possibility of false-negative outcomes due to limited sample sizes in some subgroups, aggregation bias and heterogeneity (Higgins and Thompson, 2002).

Quantitative characterization

We estimated that mechanically stimulated mammalian cells release 38.6 (95% CI: 18.2–81.8) amol ATP/cell, resulting in a 4.3-fold (95% CI: 3.8–4.8) increase in ATP above basal levels of 8.1 (95% CI: 3.9–16.6) amol ATP/cell. Intracellular ATP content was estimated to be 3 orders of magnitude higher than basal ATP levels in nucleated cells, 5.0 (95% CI: 2.6–9.5) fmol ATP/cell, and 2 orders of magnitude higher in RBCs, 0.14 (95% CI: 0.12, 0.18) fmol ATP/RBC. Study-level estimates of the absolute amount of ATP released ranged over 10 orders of magnitude, with 5 studies reporting more ATP release than can be contained within a cell, suggesting that more caution must be taken when performing ATP calibrations and measurements, and that basal, released and total ATP content should be reported to obtain relative measures. The characteristic time to half-max ATP release was strongly influenced by the recording method, yielding almost 4 times faster estimates by real-time recordings compared with estimates acquired by bulk sampling and offline measurement. This difference can potentially be explained by the diffusion time required to equilibrate the concentration within the volume of the culture medium. In this regard, the volume into which ATP is released is an important (but not routinely controlled for) determinant of the effective ATP concentration available for autocrine and paracrine signaling, including ATP-regulated ATP release (Bodin and Burnstock, 1996 Dillon et al., 2013). We found that less ATP is released in response to cyclic stimulation however, no studies reported the kinetics of ATP release in response to repeated or cyclical stimulations, even though physiological stimuli are often cyclical (Burr et al., 1996 Eyckmans et al., 2011 Fritton et al., 2000). Of interest, studies in which cell injury was assessed and detected or intentionally induced reported a tendency for higher ATP release. However, the amount of injury-related ATP release never reached amounts expected following cell destruction and was not statistically different from osmotic pressure- or FSS-induced ATP release. Thus, quantitative synthesis of basic science studies employing diverse approaches with complex endpoints is feasible and has allowed us to identify methodological variations of consequence.

Dependence on mechanical stimulus

We compared ATP release induced by 9 different types of mechanical stimulation, including stretch-related stimuli, such as substrate strain, osmotic pressure and tissue distention, and FSS and local membrane deformation. Although all types of mechanical stimulation resulted in the release of comparable amounts of ATP, we found significant differences in the kinetics of ATP release, which were much faster in response to FSS- compared with stretch-related stimuli. In addition, we found that VRAC and possibly pannexins were involved in mediating swell- and strain-induced, but not shear-induced, responses. Finally, MSAR from PKD-afflicted renal epithelia was differentially sensitive to FSS and hypotonic swelling. These results strongly suggest that mammalian cells can discriminate between different types of mechanical stimuli. Theoretical models have previously demonstrated that shear stresses induce more cell membrane deformation than stretch-related stimuli (Lynch and Fischbach, 2014 McGarry et al., 2005). Direct comparison of mechanisms involved in hypotonic pressure- and strain-induced ATP release has demonstrated that these stretch-related stimuli recruit common ATP release pathways (Li et al., 2011) however, no study has directly compared shear- and stretch-related ATP release. Thus, we recommend a direct comparison between shear- and stretch-induced cell deformation, and ATP release to be investigated in future work.

Intervention studies

We quantified the effects of pharmacological and genetic intervention for 681 combinations of cell type, mechanical stimulation and interventions. From the five main routes of ATP release [vesicular (Bodin and Burnstock, 2001 Sathe et al., 2011), pannexin (Locovei et al., 2006), connexin (Graff et al., 2000), VRAC (Pedersen et al., 1999 Qiu et al., 2014 Voss et al., 2014) and MAC (Sabirov et al., 2001 Sabirov et al., 2017)], at least 2, and often 3, were consistently implicated in the same cell type, with the exception of keratinocytes, in which all 5 pathways were studied, but only MAC was found to mediate ATP release. The involvement of multiple independent release mechanisms may confer a redundancy that ensures that cellular ATP release is robust. Alternatively, it is possible that different routes work collaboratively within the same pathway. Of interest, we demonstrated a lack of additivity in the relative contributions of main release routes in certain cell types. We found that different ATP release routes shared common intracellular signaling pathways. In particular, Rho kinases and microtubules always co-occurred with vesicular and pannexin pathways, and [Ca 2+ ]i co-occurred with all 5 routes of ATP release in 33–100% of cases. Thus, it can be hypothesized that different routes of release are functionally independent but are regulated by common intracellular signaling. At this time, this hypothesis is difficult to test experimentally, partly because pharmacological agents used to study mechanisms of MSAR suffer from extensively overlapping antagonistic profiles (Azorin et al., 2011 Li et al., 2010 Liu et al., 2008 Sauer et al., 2000 Wang et al., 2005). Targeted genetic studies (e.g. siRNA, CRISPR, animal models) are needed to further our understanding of MSAR. To date, genetic interventions have been used to study the involvement of vesicle-related vesicular nucleotide transporter (VNUT also known as SLC17A9) (Sathe et al., 2011 Sawada et al., 2008), pannexin 1 (Panx1) (Bao et al., 2004 Beckel et al., 2014 Kanjanamekanant et al., 2014 Lu et al., 2012 Seminario-Vidal et al., 2011 Woehrle et al., 2010), pannexin 2 (Panx2) (Oishi et al., 2012), connexin 40 (Cx40 also known as GJA5) (Toma et al., 2008), connexin 43 (Cx43) (Chi et al., 2014 Genetos et al., 2007 Lu et al., 2012 Luckprom et al., 2011) and connexin 45 (Cx45 also known as GJC1) (Lu et al., 2012). Recently, SWELL1 (also known as LRRC8A) was identified as the pore component of the VRAC complex (Qiu et al., 2014 Syeda et al., 2016 Voss et al., 2014), and the prostaglandin transporter PGT (encoded by Slco2a1) was recognized as the MAC (Sabirov et al., 2017). Of interest, Sana-Ur-Rehman et al. (2017) and Workman et al. (2017) have recently reported the calcium homoeostatic modulator 1 (CALHM1) as a novel mediator of MSAR in nasal epithelia and the urothelium however, it remains unclear whether CALHM1 is a direct or indirect conduit of ATP release. A comprehensive review of recent evidence supporting the role of CALHM1 in ATP release can be found elsewhere (Taruno, 2018). Nonetheless, now that the molecular identity of each main route of ATP release has been identified, genetic studies to resolve ATP release mechanisms are feasible. Thus, systematic analysis of prior data allowed us to pinpoint specific mechanistic features that warrant further experimental investigation, such as differential involvement of VRAC in FSS- and osmotic pressure-induced ATP release, and to suggest that certain ATP release mechanisms are cell type and stimulation type dependent.

Therapeutic potential

Our systematic assessment of MSAR involvement in different pathologies included data from 10 cell types/tissues from 11 pathological conditions. We have found that inflammation and injury coincided with higher ATP release from epithelial cells, which might contribute to pain commonly present in these conditions (Butrick et al., 2010 Docherty et al., 2011 Taweel and Seyam, 2015 Weinreb et al., 2014). In contrast, in hereditary and metabolic conditions, lower ATP release from RBCs was consistently reported. Thus, both MSAR inhibitory and stimulatory interventions can be of therapeutic interest. The downstream actions of MSAR are mediated by 15 members of the purinergic (P2) receptor family (Burnstock, 2014), which have been identified as valuable therapeutic targets for treatment of pain, inflammation, spinal cord injury and bladder dysfunction (North and Jarvis, 2013). There are several advantages of targeting MSAR over the P2 receptor network. First, the impact of disproportionally targeting single P2 receptors has poorly understood implications for signaling by the entire P2 receptor network. Instead, manipulating MSAR allows proportional reduction or increase in the stimulation of all P2 receptors. Second, many of the drugs used to inhibit MSAR, including mefloquine (Lee et al., 2017), carbenoxolone (Doll et al., 1965), probenecid (Li et al., 2016), flufenamic acid (Flemming and Jones, 2015), glybenclamide (Sola et al., 2015) and clodronate (Ghinoi and Brandi, 2002) [recently demonstrated to potently inhibit VNUT (Kato et al., 2017)] are already used in clinic. Although these drugs are relatively non-specific, strategies to therapeutically re-purpose them for diseases with aberrant MSAR may be considered. As for any potential therapy, unintended drug effects need to be taken into account. In this regard, our systematic approach allowed the identification of cell- and stimulus-specific effects of various pathologies. Thus, comprehensive assessment of cell type-specific mechanisms of ATP release, considered together with known pathophysiological changes, can be used to map site-specific effects of therapeutic MSAR interventions, and predict any unintended (patho)physiological consequences.


Introduction

With the healthcare burden of cardiovascular disease increasing every year, considerable research efforts are devoted to the better understanding of underlying disease pathologies and to the development and screening of novel pharmacological treatments. While there is still a significant reliance on conventional 2D cell culture platforms for these studies, it is apparent that there is an urgent need for 3D culture systems that are more representative of in vivo tissue microenvironments. Specifically, engineered heart tissues (EHTs) such as those pioneered by Zimmermann et al. and Bursac et al. have come to the forefront as a promising platform for cardiovascular research [1,2]. Unfortunately, the use of non-human cell lines in these tissues can lead to incidences of false positives or negatives, and thereby unnecessary risk to patients, due to mismatches in pharmacokinetics and physiology between species [3], and the acquisition of sufficient numbers of terminally-differentiated human primary cardiomyocytes is time-consuming and costly. The advent of human induced pluripotent stem cells (hiPSCs) has somewhat ameliorated these issues since not only are hiPSCs readily obtainable and scalable in culture, but hiPSC lines can now be generated from patients with specific disease phenotypes [4,5]. Furthermore, it has been demonstrated that hiPSC-derived cells can also be incorporated and assembled into EHTs, and that the potential is there for these humanized tissues to serve as an important research tool.

However, cardiomyocytes differentiated from hiPSCs are functionally immature and behave more like cells that are at a neonatal, rather than adult, stage of development [6]. This has severely limited the widespread adoption of EHTs for preclinical studies, as their inability to provide accurate responses to drugs or to reproduce disease phenotypes hampers their overall utility. To address this issue with cardiomyocyte maturity, various strategies have been studied in the field. For example, studies have demonstrated that preconditioning engineered cardiac tissues with specific biochemical, mechanical, and electrical stimulation regimes elicited beneficial effects on overall tissue function and maturation [[7], [8], [9]]. However, these techniques can be limited by their requirement for the integration of complex hardware and software, thereby resulting in potential difficulties with platform adaptability and in increasing experimental throughput. Alternatively, improved maturity can be potentially achieved by exploring attempts to recapitulate the myocardial extracellular matrix (ECM) that defines the architecture, signaling, and biomechanics of cardiomyocytes in vivo [10]. Naturally-derived polymers typically used to engineer 3D tissues, such as fibrin and collagen, offer good biocompatibility and biochemical similarity to in vivo systems. In particular, decellularized extracellular matrices (dECM) are able to largely preserve the tissue-specific biochemical makeup of the tissues that they are derived from [11]. The poor mechanical and electrical properties of these polymers, however, often hampers their ability to replicate the tissue microenvironment. Synthetic materials on the other hand, offer better control over their physical and chemical characteristics, but their bioactivity is often non-existent without further modification. Perhaps the most interesting exceptions are highly-electroconductive materials, such as carbon nanotubes/nanofibers [12,13], graphene derivatives [14], and gold [15], that have been found to impart beneficial effects on the growth and maturation of a variety of cell types, including cardiomyocytes, and have recently gained increasing prominence in the field of cardiac tissue engineering. Despite these advances, engineered cardiac tissue function and physiological relevance is still at suboptimal levels due to limitations with the scaffold materials and platforms that are currently available.

Here, we report on the development of hybrid hydrogel tissue engineering scaffolds that are comprised of reduced graphene oxide (rGO) dispersed within dECM, where the electrical and mechanical properties of dECM-rGO hydrogels were modulated by controlling the degree of reduction of GO to a more graphene-like sp 2 hybridized lattice structure that imparts high conductivity and mechanical strength [16]. This tunable nature of rGO made it an attractive candidate for the electroconductive component of the hydrogels, and while the use of graphene or carbon nanotubes would have likely resulted in hydrogels with even greater electroconductivities and stiffnesses, the relatively hydrophobic nature of these materials compared to rGO would lead to poor dispersion within aqueous matrices, resulting in heterogeneity in cellular response across engineered tissues. Furthermore, the residual oxygen-containing groups in rGO could induce the spontaneous adsorption of proteins and thereby act as depots for biomolecules that contribute to cell differentiation and development [17]. By leveraging the advantages offered by rGO with regards to controlling properties such as stiffness and electroconductivity while maintaining the bioactivity inherent with dECM, this new material could help advance the capability to engineer 3D human cardiac tissues that are more physiologically-representative of the native myocardium.


Acknowledgements

Experiments using Zeiss Lightsheet Z.1 were conducted at the UC-Berkeley Molecular Imaging Center (RRID:SCR_0122850), the Cancer Research Laboratory, and the Helen Wills Neuroscience Institute (HWNI), with training and assistance from Holly Aaron and Jen-Yi Lee. We thank Brian Calvi, Shigeo Hayashi, Tetsuya Kojima, Greg Beitel, Yang Hong, TRiP at Harvard Medical School (NIH/NIGMS R01-GM084947), Kyoto Stock Center, the Vienna Drosophila Resource Center (VDRC) 71 , and the Bloomington Drosophila Stock Center (NIH P40OD018537) for providing fly stocks and reagents, and Laura Mathies for cloning 10XSTAT-GAL4 with advice from Ryan Boileau, Martin Zeidler, and Erika Bach. This work was supported by NIH RO1 grants GM068675 and GM111111 to D.B. and 4R00HD088708–03 to S.J.S


Mesenchymal stromal cells (MSCs), also known as mesenchymal stem cells, have been intensely investigated for clinical applications within the last decades. However, the majority of registered clinical trials applying MSC therapy for diverse human diseases have fallen short of expectations, despite the encouraging pre-clinical outcomes in varied animal disease models. This can be attributable to inconsistent criteria for MSCs identity across studies and their inherited heterogeneity. Nowadays, with the emergence of advanced biological techniques and substantial improvements in bio-engineered materials, strategies have been developed to overcome clinical challenges in MSC application. Here in this review, we will discuss the major challenges of MSC therapies in clinical application, the factors impacting the diversity of MSCs, the potential approaches that modify MSC products with the highest therapeutic potential, and finally the usage of MSCs for COVID-19 pandemic disease.

Mesenchymal stromal cells (MSCs) are pluripotent non-hematopoietic stem cells with self-renewal capability [1] and being intensively investigated in clinical trials. Since the discovery of MSCs from bone marrow by Friedenstein in 1970s, MSCs have been isolated from various sources including muscle, umbilical cord, liver, placenta, skin, amniotic fluid, synovial membrane, and tooth root [2, 3], and tested in amounts of preclinical and clinical studies (Fig.  1 ). It is now understood that MSCs have wide-ranging physiological effects including the maintenance of tissue homeostasis and regeneration [4, 5], as well as the immunomodulatory activities suitable for therapeutic application [6]. So their indications have been expanded to graft-versus-host disease (GVHD), multiple sclerosis (MS), Crohn’s disease (CD), amyotrophic lateral sclerosis (ALS), myocardial infarction (MI), and acute respiratory distress syndrome (ARDS) [7𠄹].

Various sources of MSCs used in the registered clinical trials. MSCs isolated from bone marrow are most widely applied in clinical trials, followed by those from umbilical cord and adipose. MSCs from muscles, tooth are also used

Over 300 clinical trials of MSC therapies have been completed in patients including but not limited to degenerative or autoimmune diseases (Table ​ (Table1 1 lists some of the representative completed studies). Overall, MSCs have exhibited tolerable safety profile and demonstrated promising therapeutic benefits in some clinical settings, which led to regulatory approvals of MSCs in a few countries. In 2011, the Ministry of Food and Drug Safety (Korea FDA) approved Cartistem®, a MSC product derived from umbilical cord blood and developed by Medipost for the treatment of traumatic or degenerative osteoarthritis [10]. Thereafter, more MSC products including HeartiCellgram®, Mesoblast, TiGenix, and Stempeutics, were approved by regulatory authorities worldwide for the treatment of a variety of diseases. In the USA, Ryoncil (remestemcel-L) is promising to be the first FDA-approved GVHD treatment for children younger than 12, but is still in the stage of safety verification. The amount of clinics offering exogenous stem cell therapies has doubled from 2009 to 2014 in the USA. This boom in stem cell clinics with 351 companies putting stem cells for sale in 570 clinics in 2016 indicated the mal-practice of the MSC therapies [11]. Considering the fact that many of the applied exogenous stem cell therapies lack confirmation on safety and effectiveness from large-scale clinical trials and are even illegal, these medical mal-practices do threaten the development of MSC therapies [12].

Table 1

Some representative registered clinical trials of MSC therapies

National Heart, Lung, and Blood Institute (NHLBI)

Massachusetts General Hospital

University of California, San Francisco

University of Alabama at Birmingham

Universidad de los Andes, Chile

Stemedica Cell Technologies, Inc

Fundacion Teknon, Centro Medico Teknon, Barcelona

National Heart, Lung, and Blood Institute (NHLBI)

Johns Hopkins University Specialized Center for Cell Based Therapy

The Prince Charles Hospital

Mater Medical Research Institute

The University of Texas Health Science Center, Houston

National Heart, Lung, and Blood Institute (NHLBI)

National Heart, Lung, and Blood Institute (NHLBI)

National Institutes of Health Clinical Center (CC)

National Heart, Lung, and Blood Institute (NHLBI)

National Heart, Lung, and Blood Institute (NHLBI)

University Medical Centre Ljubljana

Blood Transfusion Centre of Slovenia

Icahn School of Medicine at Mount Sinai

National Heart, Lung, and Blood Institute (NHLBI)

In this review, we will focus on the major challenges of MSC therapies and the underlying factors leading to the failure of clinical trials. Recent advances and prospects concerning the translation of MSC techniques into clinical practices will also be discussed.


3.8.4: Extracellular Matrix of Animal Cells - Biology

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Tonicity refers to the amount of solute in the extracellular fluid, which affects osmosis and results in three possible scenarios of altering the cell's volume.

When there are equal concentrations of solute present inside and outside, the solution is isotonic. There is no net movement of water, as water will still move in and out, just in equal proportions.

However, if there is a low-solute and high-water concentration outside relative to inside, the condition is hypotonic, and water will move into the cell causing it to swell and maybe even burst.

In contrast during hypertonicity, the extracellular fluid contains more solute and less water than the inside, thus water moves out of the cell causing it to shrink.

5.7: Tonicity in Animals

The tonicity of a solution determines if a cell gains or loses water in that solution. The tonicity depends on the permeability of the cell membrane for different solutes and the concentration of nonpenetrating solutes in the solution within and outside of the cell. If a semipermeable membrane hinders the passage of some solutes but allows water to follow its concentration gradient, water moves from the side with low osmolarity (i.e., less solute) to the side with higher osmolarity (i.e., higher solute concentration). Tonicity of the extracellular fluid determines the magnitude and direction of osmosis and results in three possible conditions: hypertonicity, hypotonicity, and isotonicity.

Isotonic Solutions

In biology, the prefix &ldquoiso&rdquo means equal or being of equal measurements. When extracellular and intracellular fluid have an equal concentration of nonpenetrating solute inside and outside, the solution is isotonic. Isotonic solutions have no net movement of water. Water will still move in and out, just in equal proportions. Therefore, no change in cell volume occurs.

Hypotonic Solutions

The prefix &ldquohypo&rdquo means lower or below. Whenever there is a low concentration of nonpenetrating solute and a high concentration of water outside relative to inside, the environment is hypotonic. Water will move into the cell, causing it to swell. In animal cells, the swelling ultimately causes cells to burst and die. Freshwater is an example of a hypotonic environment. Freshwater organisms tend to have higher osmolarity (i.e., higher salt concentration) inside their cells than the surrounding body of water such as a lake or river.

Hypertonic Solutions

Conversely, the prefix &ldquohyper&rdquo means more or above. During hypertonicity, the extracellular fluid contains more solute (i.e., high osmolarity) and less water than the inside of a cell. Thus, water moves out of the cell, causing animal cells to shrink. Saltwater is an example of hypertonic extracellular fluid because it has a higher osmolarity (i.e., higher salt concentration) in contrast to most intracellular fluids.

Osmoregulation

To avoid the shrinking and swelling that occurs in hypertonic and hypotonic solutions, animal cells must have strategies to maintain osmotic balance. The process by which osmotic balance is achieved is called osmoregulation. Osmoregulatory strategies can be grouped into two categories: regulating and conforming. Osmoregulators control and maintain their internal osmotic conditions independent of environmental conditions. Conversely, osmoconformers use active and passive internal processes to mimic the osmolarity of their environment.

Many animals, including humans, are osmoregulators. For instance, fish that live in saltwater, a hypertonic environment, are able to regulate water lost to the environment by taking in copious quantities of water and frequently excreting salt out. Fish that live in freshwater mitigate the constant osmosis of water into their cells by frequent urination that releases water out of the body.

Most marine invertebrates, such as lobsters and jellyfish, are osmoconformers. Osmoconformers maintain an internal solute concentration&mdashor osmolarity&mdashequal to that of their surroundings, and so they thrive in environments without frequent fluctuations.

Vujovic, Predrag, Michael Chirillo, and Dee U. Silverthorn. &ldquoLearning (by) Osmosis: An Approach to Teaching Osmolarity and Tonicity.&rdquo Advances in Physiology Education 42, no. 4 (October 10, 2018): 626&ndash35. [Source]


References

Betts, J. G., Young, K.A., Wise, J.A., Johnson, E., Poe, B., Kruse, D.H., Korol, O., Johnson, J.E., Womble, M., DeSaix, P. (2013, April 25). Figure 4.18 Muscle tissue [digital image]. In Anatomy and Physiology (Section 4.4). OpenStax. https://openstax.org/books/anatomy-and-physiology/pages/4-4-muscle-tissue-and-motion

Betts, J. G., Young, K.A., Wise, J.A., Johnson, E., Poe, B., Kruse, D.H., Korol, O., Johnson, J.E., Womble, M., DeSaix, P. (2013, April 25). Figure 6.11 Bone cells [digital image]. In Anatomy and Physiology (Section 6.3). OpenStax. https://openstax.org/books/anatomy-and-physiology/pages/6-3-bone-structure

Blausen.com Staff. (2014). Medical gallery of Blausen Medical 2014. WikiJournal of Medicine 1 (2). DOI:10.15347/wjm/2014.010. ISSN 2002-4436.

Brainard, J/ CK-12 Foundation. (2016). Figure 3 Five subtypes of human white blood cells in the human immune system [digital image]. In CK-12 College Human Biology (Section 9.3) [online Flexbook]. CK12.org. https://www.ck12.org/book/ck-12-college-human-biology/section/9.3/

TED-Ed. (2013, May 17). Could tissue engineering mean personalized medicine? – Nina Tandon. YouTube. https://www.youtube.com/watch?v=_7TKiFRkKGY&feature=youtu.be

TED-Ed. (2013, March 15). Printing a human kidney – Anthony Atala. YouTube. https://www.youtube.com/watch?v=bX3C201O4MA&feature=youtu.be

Tissue which lines the outer surfaces of organs and blood vessels throughout the body, as well as the inner surfaces of cavities in many internal organs. An example is the epidermis, the outermost layer of the skin. There are three principal shapes of epithelial cell: squamous, columnar, and cuboidal.

One of the four basic types of tissue, connective tissue is found in between other tissues everywhere in the body, including the nervous system and generally forms a framework and support structure for body tissues and organs.

A three-dimensional network of extracellular macromolecules, such as collagen, enzymes, and glycoproteins, that provide structural and biochemical support to surrounding cells.

A soft tissue that composes muscles in animal bodies, and gives rise to muscles' ability to contract. This is opposed to other components or tissues in muscle such as tendons or perimysium.

A specialized tissue found in the central nervous system and the peripheral nervous system. It consists of neurons and supporting cells called neuroglia. The nervous system is responsible for the control of the body and the communication among its parts.

A person who donates one kidney or a portion of their liver or a part of their lung to someone who needs those organs to survive. Transplants from living donors have been extremely successful and most donors recover with very few complications.


Watch the video: Active, Passive, and Bulk Cell Transport (July 2022).


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