Growth hormone insensitivity and adipose tissue: tissue morphology and transcriptome analyses in pigs and humans.

PURPOSE: Growth hormone receptor knockout (GHR-KO) pigs have recently been developed, which serve as a large animal model of Laron syndrome (LS). GHR-KO pigs, like individuals with LS, are obese but lack some comorbidities of obesity. The purpose of this study was to examine the histological and transcriptomic phenotype of adipose tissue (AT) in GHR-KO pigs and humans with LS. METHODS: Intraabdominal (IA) and subcutaneous (SubQ) AT was collected from GHR-KO pigs and examined histologically for adipocyte size and collagen content. RNA was isolated and cDNA sequenced, and the results were analyzed to determine differentially expressed genes that were used for enrichment and pathway analysis in pig samples. For comparison, we also performed limited analyses on human AT collected from a single individual with and without LS. RESULTS: GHR-KO pigs have increased adipocyte size, while the LS AT had a trend towards an increase. Transcriptome analysis revealed 55 differentially expressed genes present in both depots of pig GHR-KO AT. Many significant terms in the enrichment analysis of the SubQ depot were associated with metabolism, while in the IA depot, IGF and longevity pathways were negatively enriched. In pathway analysis, multiple expected and novel pathways were significantly affected by genotype, i.e. KO vs. controls. When GH related gene expression was analyzed, SOCS3 and CISH showed species-specific changes. CONCLUSION: AT of GHR-KO pigs has several similarities to that of humans with LS in terms of adipocyte size and gene expression profile that help describe the depot-specific adipose phenotype of both groups.

Growth hormone receptor gene disruption.

Much of our understanding of growth hormone's (GH)'s numerous activities stems from studies utilizing GH receptor (GHR) knockout mice. More recently, the role of GH action has been examined by creating mice with tissue-specific or temporal GHR disruption. To date, 37 distinct GHR knockout mouse lines have been created. Targeted tissues include fat, liver, muscle, heart, bone, brain, macrophage, intestine, hematopoietic stem cells, pancreatic ß cells, and inducible multi-tissue "global" disruption at various ages. In this chapter, a summary of each mouse line is provided with background information on the generation of the mouse line as well as important physiological outcomes resulting from GHR gene disruption. Collectively, these mouse lines provide unique insights into GH action and have resulted in the development of new hypotheses about the functions ascribed to GH action in particular tissues.

GHR disruption in mature adult mice alters xenobiotic metabolism gene expression in the liver.

BACKGROUND: Lifelong reduction of growth hormone (GH) action extends lifespan and improves healthspan in mice. Moreover, congenital inactivating mutations of GH receptor (GHR) in mice and humans impart resistance to age-associated cancer, diabetes, and cognitive decline. To investigate the consequences of GHR disruption at an adult age, we recently ablated the GHR at 6-months of age in mature adult (6mGHRKO) mice. We found that both, male and female 6mGHRKO mice have reduced oxidative damage, with males 6mGHRKO showing improved insulin sensitivity and cancer resistance. Importantly, 6mGHRKO females have an extended lifespan compared to controls. OBJECTIVE AND METHODS: To investigate the possible mechanisms leading to health improvements, we performed RNA sequencing using livers from male and female 6mGHRKO mice and controls. RESULTS: We found that disrupting GH action at an adult age reduced the gap in liver gene expression between males and females, making gene expression between sexes more similar. However, there was still a 6-fold increase in the number of differentially expressed genes when comparing male 6mGHRKO mice vs controls than in 6mGHRKO female vs controls, suggesting that GHR ablation affects liver gene expression more in males than in females. Finally, we found that lipid metabolism and xenobiotic metabolism pathways are activated in the liver of 6mGHRKO mice. CONCLUSION: The present study shows for the first time the specific hepatic gene expression profile, cellular pathways, biological processes and molecular mechanisms that are driven by ablating GH action at a mature adult age in males and females. Importantly, these results and future studies on xenobiotic metabolism may help explain the lifespan extension seen in 6mGHRKO mice.

Increased Fibrosis in White Adipose Tissue of Male and Female bGH Transgenic Mice Appears Independent of TGF-ß Action.

Fibrosis is a pathological state caused by excess deposition of extracellular matrix proteins in a tissue. Male bovine growth hormone (bGH) transgenic mice experience metabolic dysfunction with a marked decrease in lifespan and with increased fibrosis in several tissues including white adipose tissue (WAT), which is more pronounced in the subcutaneous (Sc) depot. The current study expanded on these initial findings to evaluate WAT fibrosis in female bGH mice and the role of transforming growth factor (TGF)-ß in the development of WAT fibrosis. Our findings established that female bGH mice, like males, experience a depot-dependent increase in WAT fibrosis, and bGH mice of both sexes have elevated circulating levels of several markers of collagen turnover. Using various methods, TGF-ß signaling was found unchanged or decreased-as opposed to an expected increase-despite the marked fibrosis in WAT of bGH mice. However, acute GH treatments in vivo, in vitro, or ex vivo did elicit a modest increase in TGF-ß signaling in some experimental systems. Finally, single nucleus RNA sequencing confirmed no perturbation in TGF-ß or its receptor gene expression in any WAT cell subpopulations of Sc bGH WAT; however, a striking increase in B lymphocyte infiltration in bGH WAT was observed. Overall, these data suggest that bGH WAT fibrosis is independent of the action of TGF-ß and reveals an intriguing shift in immune cells in bGH WAT that should be further explored considering the increasing importance of B cell-mediated WAT fibrosis and pathology.

Promotion of Joint Degeneration and Chondrocyte Metabolic Dysfunction by Excessive Growth Hormone in Mice.

OBJECTIVE: Many patients with acromegaly, a hormonal disorder with excessive growth hormone (GH) production, report pain in joints. We undertook this study to characterize the joint pathology of mice with overexpression of bovine GH (bGH) or a GH receptor antagonist (GHa) and to investigate the effect of GH on regulation of chondrocyte cellular metabolism. METHODS: Knee joints from mice overexpressing bGH or GHa and wild-type (WT) control mice were examined using histology and micro-computed tomography for osteoarthritic (OA) pathologies. Additionally, cartilage from bGH mice was used for metabolomics analysis. Mouse primary chondrocytes from bGH and WT mice, with or without pegvisomant treatment, were used for quantitative polymerase chain reaction and Seahorse respirometry analyses. RESULTS: Both male and female bGH mice at ~13 months of age had increased knee joint degeneration, which was characterized by loss of cartilage structure, expansion of hypertrophic chondrocytes, synovitis, and subchondral plate thinning. The joint pathologies were also demonstrated by significantly higher Osteoarthritis Research Society International and Mankin scores in bGH mice compared to WT control mice. Metabolomics analysis revealed changes in a wide range of metabolic pathways in bGH mice, including beta-alanine metabolism, tryptophan metabolism, lysine degradation, and ascorbate and aldarate metabolism. Also, bGH chondrocytes up-regulated fatty acid oxidation and increased expression of Col10a. Joints of GHa mice were remarkably protected from developing age-associated joint degeneration, with smooth articular joint surface. CONCLUSION: This study showed that an excessive amount of GH promotes joint degeneration in mice, which was associated with chondrocyte metabolic dysfunction and hypertrophic changes, whereas antagonizing GH action through a GHa protects mice from OA development.

Growth Hormone Alters Circulating Levels of Glycine and Hydroxyproline in Mice.

Growth hormone (GH) has established effects on protein metabolism, such as increasing protein synthesis and decreasing amino acid degradation, but its effects on circulating amino acid levels are less studied. To investigate this relationship, metabolomic analyses were used to measure amino acid concentrations in plasma and feces of mice with alterations to the GH axis, namely bovine GH transgenic (bGH; increased GH action) and GH receptor knockout (GHRKO; GH resistant) mice. To determine the effects of acute GH treatment, GH-injected GH knockout (GHKO) mice were used to measure serum glycine. Furthermore, liver gene expression of glycine metabolism genes was assessed in bGH, GHRKO, and GH-injected GHKO mice. bGH mice had significantly decreased plasma glycine and increased hydroxyproline in both sexes, while GHRKO mice had increased plasma glycine in both sexes and decreased hydroxyproline in males. Glycine synthesis gene expression was decreased in bGH mice (Shmt1 in females and Shmt2 in males) and increased in GHRKO mice (Shmt2 in males). Acute GH treatment of GHKO mice caused decreased liver Shmt1 and Shmt2 expression and decreased serum glycine. In conclusion, GH alters circulating glycine and hydroxyproline levels in opposing directions, with the glycine changes at least partially driven by decreased glycine synthesis.

Long Term Weight Cycling Affects Fecal Microbiota of Mice.

SCOPE: Fighting obesity and associated comorbidities through dieting is not always sustained and results in a subsequent weight gain, a phenomenon referred to as weight cycling. Diet is among the most important factors in modifying the composition of gut microbiota. The objective of this work is to determine whether weight cycling affects the composition and the predicted function of mouse fecal bacteria on a long-term basis. METHODS AND RESULTS: Mice fed for 40 weeks with either high fat (HF), low fat (LF), or cycled diets (starting and ending by one of the two diets, and the reverse) exhibit a bacterial profile specific to each of the four groups. A higher proportion of Firmicutes and Bacteroidota phyla are observed in mice on Hf and LF diet, respectively. The proportion of functions dedicated to amino acid metabolism is higher in mice on HF or LF/HF diets, whereas the mice on LF or HF/LF diets have a higher proportion of functions involve in carbohydrate metabolism and vitamin B biosynthesis. CONCLUSION: Compared to continuous HF or LF diets, cyclic diet specifically alters the composition and function of the mouse fecal microbiota, suggesting that fight against weight gain should be considered on a long-term basis.

Disruption of Growth Hormone Receptor in Adipocytes Improves Insulin Sensitivity and Lifespan in Mice.

Growth hormone receptor knockout (GHRKO) mice have been used for 25 years to uncover some of the many actions of growth hormone (GH). Since they are extremely long-lived with enhanced insulin sensitivity and protected from multiple age-related diseases, they are often used to study healthy aging. To determine the effect that adipose tissue has on the GHRKO phenotype, our laboratory recently created and characterized adipocyte-specific GHRKO (AdGHRKO) mice, which have increased adiposity but appear healthy with enhanced insulin sensitivity. To test the hypothesis that removal of GH action in adipocytes might partially replicate the increased lifespan and healthspan observed in global GHRKO mice, we assessed adiposity, cytokines/adipokines, glucose homeostasis, frailty, and lifespan in aging AdGHRKO mice of both sexes. Our results show that disrupting the GH receptor gene in adipocytes improved insulin sensitivity at advanced age and increased lifespan in male AdGHRKO mice. AdGHRKO mice also exhibited increased fat mass, reduced circulating levels of insulin, c-peptide, adiponectin, resistin, and improved frailty scores with increased grip strength at advanced ages. Comparison of published mean lifespan data from GHRKO mice to that from AdGHRKO and muscle-specific GHRKO mice suggests that approximately 23% of lifespan extension in male GHRKO is due to GHR disruption in adipocytes vs approximately 19% in muscle. Females benefited less from GHR disruption in these 2 tissues with approximately 19% and approximately 0%, respectively. These data indicate that removal of GH's action, even in a single tissue, is sufficient for observable health benefits that promote long-term health, reduce frailty, and increase longevity.

Growth hormone receptor antagonism downregulates ATP-binding cassette transporters contributing to improved drug efficacy against melanoma and hepatocarcinoma in vivo.

Knockdown of GH receptor (GHR) in melanoma cells in vitro downregulates ATP-binding cassette-containing (ABC) transporters and sensitizes them to anti-cancer drug treatments. Here we aimed to determine whether a GHR antagonist (GHRA) could control cancer growth by sensitizing tumors to therapy through downregulation of ABC transporters in vivo. We intradermally inoculated Fluc-B16-F10 mouse melanoma cells into GHA mice, transgenic for a GHR antagonist (GHRA), and observed a marked reduction in tumor size, mass and tumoral GH signaling. Moreover, constitutive GHRA production in the transgenic mice significantly improved the response to cisplatin treatment by suppressing expression of multiple ABC transporters and sensitizing the tumors to the drug. We confirmed that presence of a GHRA and not a mere absence of GH is essential for this chemo-sensitizing effect using Fluc-B16-F10 allografts in GH knockout (GHKO) mice, where tumor growth was reduced relative to that in GH-sufficient controls but did not sensitize the tumor to cisplatin. We extended our investigation to hepatocellular carcinoma (HCC) using human HCC cells in vitro and a syngeneic mouse model of HCC with Hepa1-6 allografts in GHA mice. Gene expression analyses and drug-efflux assays confirm that blocking GH significantly suppresses the levels of ABC transporters and improves the efficacy of sorafenib towards almost complete tumor clearance. Human patient data for melanoma and HCC show that GHR RNA levels correlate with ABC transporter expression. Collectively, our results validate in vivo that combination of a GHRA with currently available anti-cancer therapies can be effective in attacking cancer drug resistance.

Musculoskeletal Effects of Altered GH Action.

Growth hormone (GH) is a peptide hormone that can signal directly through its receptor or indirectly through insulin-like growth factor 1 (IGF-1) stimulation. GH draws its name from its anabolic effects on muscle and bone but also has distinct metabolic effects in multiple tissues. In addition to its metabolic and musculoskeletal effects, GH is closely associated with aging, with levels declining as individuals age but GH action negatively correlating with lifespan. GH's effects have been studied in human conditions of GH alteration, such as acromegaly and Laron syndrome, and GH therapies have been suggested to combat aging-related musculoskeletal diseases, in part, because of the decline in GH levels with advanced age. While clinical data are inconclusive, animal models have been indispensable in understanding the underlying molecular mechanisms of GH action. This review will provide a brief overview of the musculoskeletal effects of GH, focusing on clinical and animal models.

Covert actions of growth hormone: fibrosis, cardiovascular diseases and cancer.

Since its discovery nearly a century ago, over 100,000 studies of growth hormone (GH) have investigated its structure, how it interacts with the GH receptor and its multiple actions. These include effects on growth, substrate metabolism, body composition, bone mineral density, the cardiovascular system and brain function, among many others. Recombinant human GH is approved for use to promote growth in children with GH deficiency (GHD), along with several additional clinical indications. Studies of humans and animals with altered levels of GH, from complete or partial GHD to GH excess, have revealed several covert or hidden actions of GH, such as effects on fibrosis, cardiovascular function and cancer. In this Review, we do not concentrate on the classic and controversial indications for GH therapy, nor do we cover all covert actions of GH. Instead, we stress the importance of the relationship between GH and fibrosis, and how fibrosis (or lack thereof) might be an emerging factor in both cardiovascular and cancer pathologies. We highlight clinical data from patients with acromegaly or GHD, alongside data from cellular and animal studies, to reveal novel phenotypes and molecular pathways responsible for these actions of GH in fibrosis, cardiovascular function and cancer.

Excess Growth Hormone Alters the Male Mouse Gut Microbiome in an Age-dependent Manner.

The gut microbiome has an important role in host development, metabolism, growth, and aging. Recent research points toward potential crosstalk between the gut microbiota and the growth hormone (GH)/insulin-like growth factor-1 (IGF-1) axis. Our laboratory previously showed that GH excess and deficiency are associated with an altered gut microbial composition in adult mice. Yet, no study to date has examined the influence of GH on the gut microbiome over time. Our study thus tracked the effect of excess GH action on the longitudinal changes in the gut microbial profile (ie, abundance, diversity/maturity, predictive metabolic function, and short-chain fatty acid [SCFA] levels) of bovine GH (bGH) transgenic mice at age 3, 6, and 12 months compared to littermate controls in the context of metabolism, intestinal phenotype, and premature aging. The bGH mice displayed age-dependent changes in microbial abundance, richness, and evenness. Microbial maturity was significantly explained by genotype and age. Moreover, several bacteria (ie, Lactobacillus, Lachnospiraceae, Bifidobacterium, and Faecalibaculum), predictive metabolic pathways (such as SCFA, vitamin B12, folate, menaquinol, peptidoglycan, and heme B biosynthesis), and SCFA levels (acetate, butyrate, lactate, and propionate) were consistently altered across all 3 time points, differentiating the longitudinal bGH microbiome from controls. Of note, the bGH mice also had significantly impaired intestinal fat absorption with increased fecal output. Collectively, these findings suggest that excess GH alters the gut microbiome in an age-dependent manner with distinct longitudinal microbial and predicted metabolic pathway signatures.

Assessing utility of a lifestyle-based tool in the clinical setting as a primordial prevention strategy: The Healthy Heart Score.

OBJECTIVE: To evaluate the opinions of primary health-care providers and patients regarding the use of the Healthy Heart Score 20-year cardiovascular disease risk assessment tool in the clinical setting. METHODS: Semi-structured in person or phone interviews among 20 patients with no self-reported cardiovascular disease diagnoses and 20 health-care providers in Central (Columbus) and Southeastern (Athens) regions of Ohio. The researchers independently coded transcribed interviews, discussed codes to resolve discrepancies, and agreed on common themes. RESULTS: Participants suggested ways to best utilize and improve the tool, including adding graphics for visual reference of serving size. Patients showed interest in cardiovascular disease primordial prevention and expressed willingness to take the assessment prior to seeing a health-care provider. Health-care providers said that they would recommend the assessment to their patients and would be willing to use the tool in their practice. Health-care providers stated few barriers to using the tool, yet discussed numerous challenges to successful primordial prevention. CONCLUSIONS: Our findings support the utilization of the Healthy Heart Score as a cardiovascular disease primordial prevention tool in the clinical setting. Additional research implementing the tool into the clinical setting will provide deeper insight into how the tool can impact behavior change and cardiovascular disease prevention.

Growth hormone alters gross anatomy and morphology of the small and large intestines in age- and sex-dependent manners.

PURPOSE: Growth hormone (GH) has an important role in intestinal barrier function, and abnormalities in GH action have been associated with intestinal complications. Yet, the impact of altered GH on intestinal gross anatomy and morphology remains unclear. METHODS: This study investigated the influence of GH signaling on gross anatomy, morphology, and fibrosis by characterizing the small and large intestines in male and female bovine growth hormone transgenic (bGH) mice and GH receptor gene-disrupted (GHR-/-) mice at multiple timepoints. RESULTS: The length, weight, and circumference of the small and large intestines were increased in bGH mice and decreased in GHR-/- mice across all ages. Colon circumference was significantly increased in bGH mice in a sex-dependent manner while significantly decreased in male GHR-/- mice. Villus height, crypt depth, and muscle thickness of the small intestine were generally increased in bGH mice and decreased in GHR-/- mice compared to controls with age- and sex-dependent exceptions. Colonic crypt depth and muscle thickness in bGH and GHR-/- mice were significantly altered in an age- and sex-dependent manner. Fibrosis was increased in the small intestine of bGH males at 4 months of age, but no significant differences were seen between genotypes at other timepoints. CONCLUSION: This study observed notable opposing findings in the intestinal phenotype between mouse lines with GH action positively associated with intestinal gross anatomy (i.e. length, weight, and circumference). Moreover, GH action appears to alter morphology of the small and large intestines in an age- and sex-dependent manner.

Regulation of 11ß-HSD1 by GH/IGF-1 in key metabolic tissues may contribute to metabolic disease in GH deficient patients.

Patients with growth hormone deficiency (GHD) have many clinical features in common with Cushing's syndrome (glucocorticoid excess) - notably visceral obesity, insulin resistance, muscle myopathy and increased vascular mortality. Within key metabolic tissues, 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) converts cortisone to the active glucocorticoid, cortisol (11-dehydrocorticosterone and corticosterone in rodents respectively), and thus amplifies local glucocorticoid action. We hypothesize that 11ß-HSD1 expression is negatively regulated by growth hormone (GH), and that GHD patients have elevated 11ß-HSD1 within key metabolic tissues (leading to increased intracellular cortisol generation) which contributes to the clinical features of this disease. To identify the impact of GH excess/resistance on 11ß-HSD1 in vivo, we measured mRNA expression in key metabolic tissues of giant mice expressing the bovine GH (bGH) gene, dwarf mice with a disrupted GH receptor (GHRKO) gene and mice expressing a gene encoding a GH receptor antagonist (GHA). Additionally, we assessed urine steroid markers of 11ß-HSD1 activity in both GHRKO and bGH animals. 11ß-HSD1 expression was decreased in gastrocnemius muscle (0.43-fold, p < 0.05), subcutaneous adipose (0.53-fold, p < 0.05) and epididymal adipose tissue (0.40-fold, p < 0.05), but not liver, in bGH mice compared to WT controls. This was paralleled by an increased percentage of 11-DHC (inactive glucocorticoid) present in the urine of bGH mice compared to WT controls (2.5-fold, p < 0.01) - consistent with decreased systemic 11ß-HSD1 activity. By contrast, expression of 11ß-HSD1 was increased in the liver of GHRKO (2.7-fold, p < 0.05) and GHA mice (2.0-fold, p < 0.05) compared to WT controls, but not gastrocnemius muscle, subcutaneous adipose tissue or epididymal adipose tissue. In summary, we have demonstrated a negative relationship between GH action and 11ß-HSD1 expression which appears to be tissue specific. These data provide evidence that increased intracellular cortisol production within key tissues may contribute to metabolic disease in GHD patients.

Mice with gene alterations in the GH and IGF family.

Much of our understanding of GH's action stems from animal models and the generation and characterization of genetically altered or modified mice. Manipulation of genes in the GH/IGF1 family in animals started in 1982 when the first GH transgenic mice were produced. Since then, multiple laboratories have altered mouse DNA to globally disrupt Gh, Ghr, and other genes upstream or downstream of GH or its receptor. The ability to stay current with the various genetically manipulated mouse lines within the realm of GH/IGF1 research has been daunting. As such, this review attempts to consolidate and summarize the literature related to the initial characterization of many of the known gene-manipulated mice relating to the actions of GH, PRL and IGF1. We have organized the mouse lines by modifications made to constituents of the GH/IGF1 family either upstream or downstream of GHR or to the GHR itself. Available data on the effect of altered gene expression on growth, GH/IGF1 levels, body composition, reproduction, diabetes, metabolism, cancer, and aging are summarized. For the ease of finding this information, key words are highlighted in bold throughout the main text for each mouse line and this information is summarized in Tables 1, 2, 3 and 4. Most importantly, the collective data derived from and reported for these mice have enhanced our understanding of GH action.

Growth hormone receptor gene disruption in mature-adult mice improves male insulin sensitivity and extends female lifespan.

Studies in multiple species indicate that reducing growth hormone (GH) action enhances healthy lifespan. In fact, GH receptor knockout (GHRKO) mice hold the Methuselah prize for the world's longest-lived laboratory mouse. We previously demonstrated that GHR ablation starting at puberty (1.5 months), improved insulin sensitivity and female lifespan but results in markedly reduced body size. In this study, we investigated the effects of GHR disruption in mature-adult mice at 6 months old (6mGHRKO). These mice exhibited GH resistance (reduced IGF-1 and elevated GH serum levels), increased body adiposity, reduced lean mass, and minimal effects on body length. Importantly, 6mGHRKO males have enhanced insulin sensitivity and reduced neoplasms while females exhibited increased median and maximal lifespan. Furthermore, fasting glucose and oxidative damage was reduced in females compared to males irrespective of Ghr deletion. Overall, disrupted GH action in adult mice resulted in sexual dimorphic effects suggesting that GH reduction at older ages may have gerotherapeutic effects.

Transcriptome profiling of insulin sensitive tissues from GH deficient mice following GH treatment.

PURPOSE: Most studies that have examined the transcriptional response to GH have been performed with a single tissue. Thus, the current study performed RNASeq across three insulin-sensitive tissues of GH-treated GH deficient (GHKO) mice. METHODS: GHKO mice were injected with recombinant human GH (hGH) or vehicle daily for 5 days and adipose, liver, and muscle tissues were collected 4 h after the final injection. RNA was isolated from the tissues and sequenced. Genes that were differentially expressed between GH and vehicle treatments were further analyzed. Enrichment analysis and topology-aware pathway analysis were performed. RESULTS: GHKO mice treated with hGH had expected phenotypic alterations, with increased body, fat, fluid, liver, and muscle mass, and increased serum IGF-1 and insulin. 55 Genes were differentially expressed in all three tissues, including the canonical GH targets Igf1, Igfals, and Cish. Enrichment analysis confirmed the canonical GH response in select tissues, such as cell proliferation, metabolism, and fibrosis. The JAK/STAT pathway was the only pathway significantly altered in all three tissues. CONCLUSIONS: As expected, GH caused expression changes of many known target genes, although new candidate GH targets were identified. Liver and muscle appear to be more GH sensitive than adipose tissue due to the larger number of DEG and pathways significantly altered, but adipose still has a characteristic GH response. The diversity of changes uncovered in all three tissues after 5 days of GH treatment highlights the multiplicity of GH's effects in its target tissues.