Biomedical subjects

G Schett

Publications and source records attributed to G Schett.

36 records · Page 2Linked to original sources

PubMed1999

Endothelial cytotoxicity mediated by serum antibodies to heat shock proteins of Escherichia coli and Chlamydia pneumoniae: immune reactions to heat shock proteins as a possible link between infection and atherosclerosis.

BACKGROUND: Growing evidence suggests that an immunological reaction against heat shock proteins (HSPs) may be involved in atherogenesis. Because HSPs show a high degree of amino acid sequence homology between different species, from prokaryotes to humans, we investigated the possibility of "antigenic mimicry" caused by an immunological cross-reaction between microorganisms and autoantigens. METHODS AND RESULTS: Serum antibodies against the Escherichia coli HSP (GroEL) and the 60-kDa chlamydial HSP (cHSP60) from subjects with atherosclerosis were purified by use of affinity chromatography. Western blot analyses and competitive ELISAs confirmed the cross-reaction of the eluted antibodies with human HSP60 and the bacterial counterparts. The cytotoxicity of anti-GroEL and anti-cHSP60 antibodies was determined on human endothelial cells labeled with 51Cr. A significant difference (40% versus 8%) was observed in the specific 51Cr release of heat-treated (42 degrees C for 30 minutes) and untreated cells, respectively, in the presence of these anti-HSP antibodies and complement. This effect was blocked by addition of 100 microg/mL recombinant GroEL. In addition, seropositivity against specific non-HSP60 Chlamydia pneumoniae antigens is more prominent among high-anti-HSP titer sera than low-titer sera. CONCLUSIONS: Serum antibodies against HSP65/60 cross-react with human HSP60, cHSP60, and GroEL; correlate with the presence of antibodies to C pneumoniae and endotoxin; and mediate endothelial cytotoxicity. These findings suggest that humoral immune reactions to bacterial HSPs, such as cHSP60 and GroEL, may play an important role in the process of vascular endothelial injury, which is believed to be a key event in the pathogenesis of atherosclerosis.

Adult↗

PubMed1999

Myocardial injury leads to a release of heat shock protein (hsp) 60 and a suppression of the anti-hsp65 immune response.

OBJECTIVE: While atherosclerosis is associated with high titers of autoantibodies to bacterial hsp65 crossreacting with human hsp60 (anti-hsp60 autoantibodies), myocardial infarction entails decreased humoral immune response to hsp65. We previously hypothesized that myocardial ischemia and subsequent infarction not only induce myocardial hsp60 expression, but also trigger release of myocardial hsp60 into the circulation, influencing the systemic hsp immune response via immune complex formation. METHODS: In the present study, organ culture of rat hearts under circulatory arrest provided a model of myocardiocyte injury due to ischemia. RESULTS: Reperfusion of ischemic hearts confirmed the occurrence of myocardial injury by a rise of heart enzymes. Myocardial hsp60 expression was induced up to threefold in response to ischemia, and most of hsp60 expression was localized to the muscle fibers. Analysis of coronary eluate revealed release of hsp60 from myocardium. In addition, hsp60-containing, but not hsp60-free, coronary eluate was recognized by anti-hsp65 serum antibodies and induced proliferation of hsp65-specific T cells. When hsp60-containing coronary eluate was reinjected into an hsp65-primed rat, both humoral and cellular hsp65-immune responses were strongly downregulated. CONCLUSION: Our findings demonstrate the release of highly immunogenic and crossreactive hsp60 into the circulation in response to myocardial ischemia and myocardiocyte injury.

Animals↗

PubMed1999

Activation of Fas inhibits heat-induced activation of HSF1 and up-regulation of hsp70.

Activation of heat shock factor (HSF) 1-DNA binding and inducible heat shock protein (hsp) 70 (also called hsp72) expression enables cells to resist various forms of stress and survive. Fas, a membrane-bound protein, is a central proapoptotic factor; its activation leads to a cascade of events, resulting in programmed cell death. These two mechanisms with contradictory functions, promoting either cell survival or death, were examined for their potential to inhibit each other's activation. Induction of FAS-mediated signaling was followed by a rapid decrease in HSF1-DNA binding and inducible hsp70 expression. Inhibition of HSF1-DNA binding was demonstrated to be based on absent hyperphosphorylation of HSF1 during FAS signaling. These effects of FAS activation on the HSF1/hsp70 stress response were blocked by ICE (caspase 1) inhibitors, suggesting an ICE-mediated process. Furthermore, inhibition of HSF1/hsp70 was accompanied by an increase in apoptosis rates from 20% to 50% in response to heat stress. When analyzing the effects of HSF1/hsp70 activation on Fas-mediated apoptosis, protection from apoptosis was seen in cells with induced hsp70 protein levels, but not in cells that were just induced for HSF1-DNA binding. Thus, we conclude that inhibition of HSF1/hsp70 stress response during Fas-mediated apoptosis and vice versa may facilitate a cell to pass a previously chosen pathway, stress resistance or apoptosis, without the influence of inhibitory signals.

Apoptosis↗

PubMed1998

Enhanced expression of heat shock protein 70 (hsp70) and heat shock factor 1 (HSF1) activation in rheumatoid arthritis synovial tissue. Differential regulation of hsp70 expression and hsf1 activation in synovial fibroblasts by proinflammatory cytokines, shear stress, and antiinflammatory drugs.

Heat shock proteins (hsp) have been repeatedly implicated to participate in the pathogenesis of rheumatoid arthritis (RA). Herein, we investigated the regulation of synovial hsp70 expression by analyzing the DNA-binding activity of heat shock transcription factor 1 (HSF1) as well as inducible hsp70 expression. Experiments were performed both on synovial tissue and on synovial fibroblast-like cells (SFC). Gel mobility shift analysis revealed increased HSF1 activation, and Western blotting and immunohistochemistry revealed increased hsp70 expression in RA synovial tissue, but not in synovial tissue derived from patients with osteoarthritis. Proinflammatory cytokines (TNF-alpha, IL-1alpha, IL-6), but not IFN-gamma or TGF-beta, induced activation of HSF1-DNA binding and hsp70 expression in cultivated SFC. Activation of HSF1 in SFC was accompanied by hyperphosphorylation and nuclear translocation of HSF1. Furthermore, shear stress also induced a complete heat shock response in cultivated synovial cells. In contrast, nonsteroidal antiinflammatory drugs triggered only an incomplete heat shock response, with HSF1 activation but not hsp70 induction, whereas steroids and immunosuppressive drugs did not affect the heat shock response at all. In summary, these data suggest that induction of hsp70 expression in rheumatoid synovial tissue is based on transcriptional activation of HSF1 due to the presence of proinflammatory cytokines (and possibly also shear stress).

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1998

Nuclear antigen histone H1 is primarily involved in lupus erythematosus cell formation.

OBJECTIVE: To elucidate the nature of the antigen reactive with the "lupus erythematosus (LE) cell factor," the autoantibody involved in the LE cell phenomenon. METHODS: Serum samples from systemic lupus erythematosus (SLE) patients who were positive for the LE cell phenomenon (LEc+) and SLE patients who were negative for the LE cell phenomenon (LEc-) were used to characterize the nuclear antigen bound by the LE cell factor, by immunoblotting and immunoprecipitation techniques. RESULTS: All LEc+ sera, but none of the LEc- sera, uniformly reacted with a double band of MW approximately 30 kd in nuclear extracts. Depletion of nuclear protein extracts of antigens bound by pooled LEc- serum allowed precipitation of a low molecular weight protein by pooled LEc+ serum. This protein was able to block LE cell formation by LEc+ serum. Based on its reactivity with antihistone antibody and an electrophoretic mobility identical with that of precipitated and purified histone H1, this protein was identified as histone H1. Moreover, all LEc+ sera, but none of the LEc- sera, reacted with purified histone H1 by immunoblotting, whereas other histones were reactive with both types of sera. In addition, purified histone H1, but none of the other histones, strongly inhibited the induction of LE cells by LEc+ serum. CONCLUSION: Histone H1 represents the major antigenic component recognized by the LE cell factor. Thus, the LE cell phenomenon appears to be due primarily to anti-histone H1 reactivity.

Adult↗

PubMed1998

Abundance of platelet-derived growth factors (PDGFs), PDGF receptors and activation of mitogen-activated protein kinases in brain decline with age.

Platelet-derived growth factors (PDGFs) specifically bind to PDGF receptors (PDGFRs), resulting in their activation via autophosphorylation and subsequent triggering of a cascade of phosphorylation events that include mitogen-activated protein (MAP) kinases. Most of our knowledge concerning MAP kinase activation comes from studies of cultured cells in vitro, and little is known about their activation in vivo. In the present study, we determined PDGF and PDGFR levels and MAP kinase activities, including extracellular signal-regulated protein kinases (ERK) and c-Jun NH2-terminal protein kinases (JNK) or stress-activated protein kinases (SAPK) in brain of young and older mice. Both PDGF and PDGFR proteins were most abundant in protein extracts from brain (cerebral cortex) among tissues of heart, liver, spleen, lung and kidney, as determined by Western blot analysis. PDGFR proteins in brain differed significantly between young (1 or 8 weeks) and older (14 months) mice and PDGFR phosphorylation was seen in all age groups examined by a specific antibody against phosphotyrosine. The highest activity ERK2 was also observed in brain tissues, and this activity declined with age, although ERK1 and ERK2 protein levels were not significantly altered during development and aging. Furthermore, the activity and amount of JNK/SAPK proteins were the most abundant in brain tissues and did not change with age. Thus, our findings demonstrate that the highest levels of PDGFs and PDGFRs existed in brain, and constitutive activation of MAP kinases declined with age, suggesting that signal pathways mediated by PDGF-MAP kinase cascades are important components in coordinating growth and differentiation of neurone and glial cells during development and aging.

Aging↗

PubMed1998

Endocarditis and aortal embolization caused by Aspergillus terreus in a patient with acute lymphoblastic leukemia in remission: diagnosis by peripheral-blood culture.

Disseminated infection with Aspergillus terreus is a rare disease that affects only the immunocompromised host. We report a case of systemic infection with A. terreus resulting in endocarditis, aortic embolization, and splenic infarction in a patient with acute lymphoblastic leukemia. Diagnosis through peripheral blood culture, lack of pulmonary involvement, and onset of disease during complete remission from leukemia constitute uncommon features of this case.

Aorta, Abdominal↗

PubMed1997

Macrophage-lysis mediated by autoantibodies to heat shock protein 65/60.

Macrophages in atherosclerotic lesions have been shown to express high amounts of heat shock protein 60 (hsp60), a highly conserved protein. Patients with atherosclerosis have high titers of anti-hsp65/60 antibodies (Ab) recognizing macrophages in the lesions. To elucidate the role of anti-hsp65/60 Ab in macrophage cytotoxicity, human high titer serum and purified anti-hsp65/60 Ab were tested on in vitro heat-stressed cells of a human macrophage cell line (U937) and macrophages derived from peripheral blood. Application of heat stress at 42 degrees C for 30 min resulted in marked upregulation of hsp60 mRNA, followed by increased protein expression as determined by Northern blot and FACS-analysis, respectively. Compared to unstressed cells, high titer serum and anti-hsp65/60 Ab preferentially bound to the surface of stressed U937 macrophages, but not control antibodies. Furthermore, high titer serum and anti-hsp65/60 Ab exerted significant (P < 0.01) complement-mediated cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) on stressed 51Cr-labelled U937 and peripheral blood derived macrophages. Thus, macrophages expressing hsp60 can be lysed by autoantibodies against hsp65/60, which may contribute to cell death in atherosclerotic plaques in vivo.

Antibody-Dependent Cell Cytotoxicity↗

PubMed1997

Salivary anti-hsp65 antibodies as a diagnostic marker for gingivitis and a possible link to atherosclerosis.

Levels of specific salivary IgA antibodies against mycobacterial heat shock protein (hsp) 65 are significantly increased in patients with gingivitis when compared to clinically healthy subjects. The process of identifying the hsp65 epitopes recognized by the salivary antibodies, binding to overlapping 15-mer-hsp65 peptides, was assessed. Time-resolved fluorescence immunoassays using 15-mer overlapping peptides spanning the whole hsp65 molecule revealed six distinct sequences recognized by anti-hsp65 IgA antibodies. Due to the high degree of sequence homology between mycobacterial hsp65, cognates of the hsp60 family of oral bacterial flora and human hsp60, these six epitopes may serve as cross-reactive autoantigens in certain circumstances in vivo and could incite an autoimmune response that contributes to the initiation of gingivitis.

Adult↗

PubMed1997

Epitope specificity of anti-heat shock protein 65/60 serum antibodies in atherosclerosis.

Levels of specific antibodies (Ab) against mycobacterial and human heat shock protein (hsp) 65/60 are increased in the sera of patients with atherosclerotic lesions and have been demonstrated to be capable of mediating endothelial cytotoxicity. To clarify the antigen epitopes recognized by these serum Abs, Ab binding to hsp65 deletion mutants (Dms), as well as to overlapping 15-mer and 8-mer hsp65 peptides, was assessed. Western blotting of hsp65 Dms indicated the presence of at least one epitope between amino acid (aa) residues 171 and 276, recognized by both high-titer sera and affinity-purified anti-hsp65/60 Ab. Fluorescence immunoassays using 53 15-mer peptides and Pin ELISA using 526 7-mer peptides demonstrated three distinct, conserved sequences with high affinity to high-titer sera and purified anti-hsp65/60 Ab. Two N-terminal sequences, aa 97-109 and aa 179-187, and one C-terminal sequence, aa 504-512, were identified. These three epitopes recognized by anti-hsp65/60 Ab may serve as autoantigens in certain circumstances in vivo. This phenomenon could contribute to the initiation of atherosclerosis by an autoimmune reaction.

Amino Acid Sequence↗

PubMed1997

Co-expression of ICAM-1, VCAM-1, ELAM-1 and Hsp60 in human arterial and venous endothelial cells in response to cytokines and oxidized low-density lipoproteins.

T-cells and monocytes are the first cells infiltrating the arterial intima during the early stages of atherogenesis. Recently our laboratory has provided evidence that T-cells isolated from atherosclerotic intima reacts against heat shock protein 60 (Hsp60). Transmigration of activated T-cells into the intima is mediated by adhesion molecules (ICAM-1; VCAM-1; ELAM-1) expressed on activated endothelial cells. Here we studied the potential of cytokines (TNF-alpha, IFN-gamma, IL-1). Escherichia coli lipopolysaccharide (LPS), native and oxidized low-density lipoprotein (LDL; oxLDL) and high temperature to induce adhesion molecules as well as Hsp60 and Hsp70 expression in human endothelial cells (EC). On Northern blots, a strong signal for ICAM-1, VCAM-1 and ELAM-1 was detected after 4 h, which thereafter declined, but did not reach the basal level of untreated control cells. Heat shock induced the expression of Hsp60 and Hsp70 but not of adhesion molecules. EC were cultivated in serum-free medium, which led to the expression of adhesion molecule transcripts. Addition of LDL or oxLDL to these ECs did not alter the expression of these transcripts. The production of adhesion molecule proteins was analysed by flow cytometry. In human venous endothelial cells (HVEC) and human arterial endothelial cells (HAEC) ICAM-1 and VCAM-1 production was permanently highly induced, whereas the high level of ELAM-1 production at 4 h disappeared after 24 h. Furthermore, only HAEC, but not HVEC, produced ICAM-1, VCAM-1 and ELAM-1 after stress by moderately and highly oxLDL. LDL and oxLDL did not induce the production of Hsp60 and Hsp70. The present study demonstrates the co-expression of Hsp60 and adhesion molecules in arterial and venous EC in response to cytokine and LPS exposure, and that oxLDL is an efficient inducer of adhesion molecules in arterial EC and not in venous EC. These features provide the prerequisites for a cellular immune reaction against Hsp60 expressed by stressed EC in the initial stages of atherosclerosis.

Antigens, CD↗

PubMed1996

Changes of serum antibodies to heat-shock protein 65 in coronary heart disease and acute myocardial infarction.

Accumulating evidence indicates the involvement of heat shock proteins (hsp), a family of stress-inducible proteins, in atherosclerosis. For carotid atherosclerosis an association with an increase in hsp65 antibodies has been demonstrated. To investigate whether such antibodies are also associated with coronary heart disease (CHD) and acute myocardial infarction (MI), an age- and sex-matched study with patients suffering from CHD (n = 114) and MI (n = 89) and healthy controls (n = 76) was performed. All study participants (n = 279) were consecutively recruited according to typical diagnostic criteria. Determination of antibody titres to hsp65 was performed by an enzyme-linked immunosorbent assay (ELISA). Hsp65 antibody titres in CHD showed a significant increase compared to the healthy control group (P = 0.029), however, hsp65 antibody titres were found to be significantly lower in acute MI, compared to CHD (P = 0.005). Alteration in hsp65 antibody titres showed no correlation to established cardiovascular risk factors, e.g. serum total cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides, blood pressure, smoking, alcohol intake and body weight. In conclusion, serum concentrations of hsp65 antibodies were elevated independently in coronary heart diseases and declined in patients with acute myocardial infarction, indicating a possible involvement of the antibodies in the pathogenesis of this disease.

Aged↗

PubMed1996

Regression of arteriosclerotic lesions induced by immunization with heat shock protein 65-containing material in normocholesterolemic, but not hypercholesterolemic, rabbits.

Previous studies in our laboratory have shown that arteriosclerotic changes can be induced in normocholesterolemic rabbits by immunization with mycobacterial heat shock protein 65 (hsp 65). To investigate the possible regression of such vascular lesions, 63 male New Zealand White rabbits were treated either by triple immunization with fortified Freund's complete adjuvant containing 5 mg/ml Mycobacterium tuberculosis, a hsp 65-rich material, by administration of a 0.2% cholesterol-rich diet only or by a combination of both immunization and cholesterol-rich diet. Sixteen weeks after the first immunization, half of the animals of each group were sacrificed, and as expected arteriosclerotic lesions in the intima of the aortic arch were found in 8 of 10 immunized animals. The remaining animals were sacrificed 16 weeks thereafter, having been maintained on a normal, non-cholesterol-enriched diet from week 16 to 32. Only 3 of 10 rabbits immunized showed moderate lesions in their aortae 32 weeks after the first immunization. On the other hand, atherosclerotic lesions induced by cholesterol-rich diet, or by immunization plus cholesterol-rich diet, showed no significant regression between 16 and 32 weeks. In conclusion, the early inflammatory stages of arteriosclerotic lesions induced by immunization with hsp 65 can regress in the absence of additional risk factors for atherosclerosis, such as a cholesterol rich diet.

Animals↗

PubMed1995

Is atherosclerosis an immunologically mediated disease?

In contrast to general beliefs, recent data from different laboratories have provided evidence that the first stages of atherosclerosis are of an inflammatory nature. Here, Georg Wick and colleagues suggest that an autoimmune reaction against heat shock protein 60 (Hsp60), expressed by endothelial cells in areas that are subject to increased haemodynamic stress, is the initiating event in atherogenesis. Humoral and T-cell-mediated immune responses against Hsp60 have both been demonstrated early in disease. This inflammatory stage, which is reversible and has even been found in children, may progress into fully developed atherosclerotic lesions, displaying all the classical pathohistological and functional consequences, if additional risk factors such as high blood cholesterol levels, smoking and obesity, are present.

Animals↗

PubMed1995

Autoantibodies against heat shock protein 60 mediate endothelial cytotoxicity.

Stress or heat shock proteins (hsp) are a family of approximately two dozen proteins with a high degree of amino acid sequence homology between different species, ranging from prokaryotes to humans, and are representative of a generalized response to environmental and metabolic stressors. Our previous studies showed increased expression of human hsp60 on endothelial cells of arterial intima with atherosclerotic lesions, and elevated levels of serum antibodies (Ab) against hsp65/60 in subjects with carotid atherosclerosis. To investigate the possible involvement of anti-hsp65/60 Ab in endothelial injury, specific hsp-Ab were isolated from human high titer sera by affinity chromatography and probed on heat-shock human umbilical vein endothelial cells. Purified human anti-hsp65/60 Ab reacted specifically with mycobacterial hsp65, human hsp60, and a 60-kD protein band of heat-shocked endothelial cells. High levels of hsp60 mRNA expression in endothelial cells were found between 4 and 12 h after 30 min treatment at 42 degrees C. In immunofluorescence tests, positive staining of heat-stressed endothelial cells was observed not only in the cytoplasm but also on the cell surface. Furthermore, only heat-stressed, but not untreated, Cr-labeled endothelial cells were lysed by anti-hsp65/60 Ab in the presence of complement (complement-mediated cytotoxicity) or peripheral blood mononuclear cells (antibody-dependent cellular cytotoxicity). Control Abs, including human anti-hsp65/60 low titer antiserum, human Ig fraction deprived of hsp65/60 Ab, and mAbs to Factor VIII, alpha-actin, hsp70, and CD3 showed no cytotoxic effect. In conclusion, human serum anti-hsp65 antibodies act as autoantibodies reacting with hsp60 on stressed endothelial cells and are able to mediate endothelial cytotoxicity. Thus, a humoral immune reaction to hsp60 may play an important role in the pathogenesis of atherosclerosis.

Aged↗

PubMed1995

Atherosclerosis as an autoimmune condition.

Atherosclerosis is a multifactorial vascular disorder responsible for the highest rate of mortality in the western world. During the last decades, research on this disease has primarily focused on the role of lipids, which are essential to the formation of lesions in the vascular intima that ultimately leads to clinically apparent atherosclerotic plaques. More recently, several anecdotal findings have indicated the possible involvement of the immune system in the process of atherogenesis. In particular, the appearance of immunocompetent cells as well as humoral antibodies in the intima in the early stages of disease development supports the view of an inflammatory component in this disorder. In addition to the search for lipid-associated antigens that might entail full-blown atherosclerosis, other candidate antigens capable of inducing an immune response in the vascular wall have also been explored. Within the probable group of antigens for immune responsiveness, heat shock protein (hsp) 60/65 became a serious candidate, upon observation that immunization of rabbits with this protein led to arteriosclerotic changes of the aortic intima. In the last few years we have established this rabbit model for immunologic investigations of atherosclerosis and, in parallel, examined the pathogenesis of human atherosclerosis with regard to hsp 60/65 immune reactivity. Currently available data point to an autoimmune induction of early inflammatory arteriosclerotic changes triggered by a cellular and humoral immune reaction to stress-induced hsp 60-expressing areas of the endothelial cells.

Animals↗

PubMed1994

Surface staining and cytotoxic activity of heat-shock protein 60 antibody in stressed aortic endothelial cells.

Heat-shock protein (hsp) expression can be induced by high temperature, exposure to cytokines or oxygen radicals, ischemia, hemodynamic overload, or viral infections. To determine whether surface expression of hsp60 occurs in aortic endothelial cells stressed by high temperature or cytokines, cells from rat aortas were cultivated and stained with several types of monoclonal antibodies against hsp60. Other antibodies, eg, those against intercellular adhesion molecule-1 (ICAM-1), or immune response-associated antigens were also used as controls. Positive staining of endothelial cells on the surface and in the cytoplasm was observed after pretreatment of the cells with cytokine-containing medium, tumor necrosis factor-alpha (TNF-alpha), or interleukin-1 alpha and labeling with a specific monoclonal antibody against hsp60 (II-13). Fluorescence-activated cell sorter analyses showed that over 80% of living endothelial cells stressed by cytokine-containing medium, by TNF-alpha, or at 42 degrees C, but not by interleukin-1 alpha, were positively surface stained with this antibody. Increased intensity of immunostaining with antibodies to ICAM-1 and immune response-associated antigen was also seen on the cytokine-stressed endothelial cells. Furthermore, when TNF-alpha stimulated endothelial cells labeled with 51Cr were incubated with antibody II-13 in the presence of complement, significant lysis occurred. In summary, endothelial cells stressed by high temperature or certain cytokines, eg, TNF-alpha, express hsp60 in the cytoplasm and on their surfaces, and these cells were susceptible to complement-dependent lysis by hsp60-specific antibody. These observations may be significant for elucidating the mechanisms of the involvement of immune reactions to hsp65/60 in initiating atherosclerosis.

Animals↗

PubMedDate not supplied

Role of heat shock protein 65/60 in the pathogenesis of atherosclerosis.

Investigations in rabbits and humans have provided experimental evidence that autoimmune reactions play a major role in the initial stages of the development of atherosclerosis. These involve the infiltration of the arterial intima with T cells reacting with heat shock protein (hsp) 65/60 and the occurrence of anti-hsp 65/60 antibodies. This early immunologically mediated stage of atherosclerosis is still reversible but if additional risk factors, such as high cholesterol levels, come into effect, severe mostly irreversible lesions develop.

Animals↗