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Gultom, et al. Proving Spike Protein Endothelial Disease: Evidence for One of My Earliest SARS-CoV-2 Hypotheses
A preprint published this week provides conclusive evidence that SPED is real – and likely responsible for Long COVID.
WALTER M CHESNUT
OCT 24

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SARS-CoV-2 spike induces a procoagulant state of the human endothelium. SARS-CoV-2 spike and TNF-a-treated HPMC and HAoEC were perfused with recalcified citrated human plasma spiked with fluorescently labeled fibrinogen. Representative images of clot formation HPMC and HAoEC 24h and 96h post-treatment (a). Time to clot formation was determined from the time series imaging, defined as the time when complete occlusion of the channel and the formation of saturated fluorescence signal from the fluorescently labeled fibrinogen were observed. Clotting time for both ECs at 24h (b) and 96h (c) after treatment. One-way ANOVA with multiple comparisons was used for statistical analysis.

A preprint was published online this week that shows the Spike Protein of SARS-CoV-2 induces a diseased state of the endothelium, The constellation of findings precisely describes what I have called SPED (Spike Protein Endothelial Disease). It was four years ago that I hypothesized the Spike Protein would induce chronic inflammation of the endothelium with activation of coagulation cascades and high levels of cellular adhesion molecules.

The paper reads as a summary of my findings over the past four years:

In line with previous findings, we showed that spike-treated ECs express high levels of cellular adhesion markers. Increased ICAM1 expression on ECs mediates the recruitment and attachment of leucocytes and neutrophil extracellular trap (NET) formation, as well as a prothrombotic state of the endothelium. Indeed, we showed that treatment with SARS-CoV-2 spike directly triggered leukocyte adhesion and increased the procoagulant state of the ECs. Elevated ICAM1 in plasma, which could be released by the damaged endothelium, is also positively correlated with disease severity as has been observed in COVID-19 patients [51], [52]. Moreover, elevated ICAM1 and other EC adhesion molecules associated with disease severity have been described in chronic cardiovascular diseases including atherosclerosis and coronary heart disease [53], [54]. Our results also show that the expression of ICAM1 seems to persist beyond the presence of SARS-CoV-2 spike, suggesting a state of sustained inflammation of the ECs. Similarly, several studies have shown increased levels of ICAM1 in serum of patients recovered from COVID-19 [35], [55], [56]. The circulating ICAM1, which could originate from damaged endothelium, may contribute to prolonged inflammation even in recovered and no longer infectious COVID-19 patients, indicating the involvement of the endothelium in PASC.

Our results showed similar profiles of chemokine expression due to SARS-CoV-2 spike activation on human EC to those observed in COVID-19 patients [30], [31]. Several studies described that elevated IL-1β, IL-6, IL-8, IL-17 in plasma is associated with disease severity in COVID-19 patients [57], [58], [59], [60], [61]. Therefore, ECs may play a significant role in the production of various inflammatory cytokines and chemokines contributing to the cytokine storm and excessive inflammatory response, exacerbating the disease in severe COVID-19 patients [58], [62]. Expression of IL-1β, CXCL1, CXCL8, and CCL20 could contribute to neutrophil recruitment to the surface of the endothelium, leading to NET-formation and immunothrombosis [60], [61], [63], [64], [65]. Chemokines such as CCL8, CXCL2, and CXCL10 can lead to recruitment of monocytes and macrophages to the activated ECs [66], [67], [68]. The increased expression of CCL2 also contributes to the amplification of monocyte and macrophage activation [69]. Recruitment of immune cells to the surface of spike-activated EC can lead to infiltration of inflammatory cells and further damage to the surrounding tissue, which can happen independently of an active infection and in different anatomical regions. Different chemokine expression levels and dynamics over time between HPMC and HAoEC suggest a possible EC origin-specific response. Our RNAseq analysis further highlights the distinct transcriptomic signatures, and the pathways associated with SARS-CoV-2 spike activation of HPMC and HAoEC. It is therefore necessary to characterize organ-specific vascular responses from organs that are also affected by COVID-19, such as brain and kidneys.

Our RNAseq data further highlight that SARS-CoV-2 spike alone can trigger an array of pathogen-associated responses, induction of robust proinflammatory states, alteration of EC development, and apoptosis, likely associated with the observed thrombo-inflammatory symptoms in COVID-19 patients [70], [71]. Moreover, prolonged expression of genes associated with proinflammatory pathways and apoptosis could induce persistent endothelial dysfunction and damage. The observed prolonged increase of adhesion molecules and antigen presentation could lengthen the recruitment of immune cells and mediate EC interaction with CD8 + and CD4 + T lymphocytes [72], [73]. In addition, the transcriptomic analysis also showed a prolonged disruption of the regulation of the complement- and coagulation cascades, reflecting a possible sustained prothrombotic state and increased cardiovascular complications after COVID-19 infection. It is worth noting that we did not see significant changes in the leucocyte binding and clotting time at a later time point in vitro, which could be due to the limitation in the assay sensitivity in our model. It is, therefore, essential to validate the long-term changes due to SARS-CoV-2 spike activation in EC in a more extensive study, for instance, in animal models or clinical studies involving convalescent COVID-19 patients. In addition, future studies should consider evaluating the consequences of EC activation by SARS-CoV-2 beyond the indicated time points, as well as the vascular inflammatory effects of other SARS-COV-2 spike variants.

In summary, our results provided a detailed and comprehensive characterization of the vascular inflammatory effects of SARS-CoV-2. We showed that the endothelium plays an essential role in determining the outcome of COVID-19 infection, such as vascular inflammation and systemic organ damage during and possibly beyond the acute infection phase. Therapeutic strategies should also consider the extent of SARS-CoV-2 inflammatory effects on the vascular endothelium. Treatments directed to EC protection and prevention of endothelial damage might be essential in the prevention and management of the post-sequelae effect of COVID-19.

Sustained vascular inflammatory effects of SARS-CoV-2 spike protein on human endothelial cells
https://www.researchsquare.com/article/rs-5003230/v1

I take no joy in having been correct. However, I am thankful that, in having been correct, those who have read my work from the beginning have always known that protecting the endothelium from the Spike Protein has been paramount.

Thank you, as always. I cannot do this without your support. My research is funded only by Paid Subscription to this Substack and Direct Donation. Currently (10/24/24), of the 13.9K Subscribers to this Substack, only 364 are Paid Subscribers. Please email me for addresses if you would like to donate Crypto. If you are comfortably able to become a Paid Subscriber, please consider doing so. Or, please donate directly at https://wmcresearch.org/donate/Gultom等人。 证明穗蛋白内皮疾病：本周发布的最早SARS-COV-2假设之一的证据提供了SPED是真实的，并且可能对长Covid负责的确定证据提供了确凿的证据。 沃尔特M Chesnut Oct 24在APP SARS-COV-2穗中阅读诱导人体内皮的促进状态。 SARS-COV-2穗和TNF-A处理的HPMC和HAOEC灌注与荧光标记的纤维蛋白原掺入的重新计算的柠檬化人血浆。 凝块形成的代表性图像HPMC和HAOEC 24H和96H后治疗后（A）。 从时间序列成像确定了凝块形成的时间，定义为在通道完全闭塞的时间和来自荧光标记的纤维蛋白原的饱和荧光信号的形成。 治疗后24小时（b）和96h（c）的凝血时间。 具有多种比较的单向ANOVA用于统计分析。 本周在线公布了一份预印刷，表明SARS-COV-2的尖峰蛋白诱导内皮的患病状态，结果精确地描述了所谓的SPED（穗蛋白内皮疾病）。 这四年前，我假设穗蛋白质会诱导内皮慢性炎症，并激活凝血级联和高水平的细胞粘附分子。 本文在过去四年中读取了我的发现：符合以前的发现，我们展示了穗治疗的ECS表达高水平的细胞粘附标记。 ECS对ICAM1表达的增加介导白细胞和中性粒细胞细胞外陷阱（净）形成的招募和附着，以及内皮的普癌态。 实际上，我们表明，用SARS-COV-2穗的治疗直接引发白细胞粘附并增加了EC的促凝血状态。 血浆中升高的ICAM1，可通过受损内皮释放，其与Covid-19患者中观察到的疾病严重程度也与疾病严重程度呈正相关[51]，[52]。 此外，已经描述了在包括动脉粥样硬化和冠心病的慢性心血管疾病中描述了与疾病严重程度相关的升高的ICAM1和与疾病严重性相关的EC粘附分子[53]，[54]。 我们的研究结果还表明，ICAM1的表达似乎持续超出SARS-COV-2穗的存在，表明ECS的持续炎症状态。 类似地，几项研究显示了从Covid-19 [35]，[55]，[56]中恢复的患者血清中ICAM1水平的增加。 循环的ICAM1，它可能来自受损内皮，即使在恢复和不再发生感染性Covid-19患者中，也可能导致延长的炎症，表明内皮患者在PASC中的参与。 我们的结果表明，由于SARS-COV-2在Covid-19患者中观察到的人EC上的SARS-COV-2 Spike Spections，趋化因子表达类似的曲线表达曲线[30]，[31]。 若干研究描述了血浆中升高的IL-1β，IL-6，IL-8，IL-17与Covid-19患者的疾病严重程度相关[57]，[58]，[59]，[60]，[61]。 因此，ECS可能在各种炎性细胞因子和趋化因子的生产中发挥重要作用，有助于细胞因子风暴和过度炎症反应，加剧严重的Covid-19患者中的疾病[58]，[62]。 IL-1β，CXCL1，CXCL8和CCL20的表达可以有助于中性粒细胞募集到内皮表面，导致净形成和免疫蛋白化[60]，[61]，[63]，[ 趋化因子如CCL8，CXCL2和CXCL10可以导致单核细胞和巨噬细胞募集到活性的ECS [66]，[67]，[68]。 CCL2的增加的表达也有助于单核细胞和巨噬细胞活化的扩增[69]。 对穗活性EC表面的免疫细胞募集可导致炎性细胞的渗透，并进一步损害周围组织，这可以独立于活性感染和不同的解剖区域发生。 HPMC和HAOEC之间的不同趋化因子表达水平和动态随着时间的推移，提出了可能的EC起源特异性响应。 我们的RNAseQ分析进一步突出了不同的转录组种签名，以及与HPMC和HAOEC的SARS-COV-2尖峰激活相关的途径。 因此，有必要表征来自Covid-19影响的器官的器官特异性血管反应，例如大脑和肾脏。 我们的RNAseq数据进一步突出显示SARS-COV-2 Spike独自可以触发一系列病原体相关的响应，诱导培养的促炎状态，EC开发的改变和凋亡，可能与观察到的血栓炎症症状相关 此外，与促炎途径和细胞凋亡相关的基因的延长表达可以诱导持续的内皮功能障碍和损伤。 观察到的粘附分子和抗原呈现的长期增加可以延长免疫细胞的募集，并介导与CD8 +和CD4 + T淋巴细胞的EC相互作用[72]，[73]。 此外，转录组分析还表明，在Covid-19感染后，反映了互补和凝血级联调节的延长破坏，反映了可能的持续癌细胞和增加的心血管并发症。 值得注意的是，在体外的稍后时间点，我们没有看到白细胞结合和凝血时间的显着变化，这可能是由于我们模型中的测定敏感性的限制。 因此，在更广泛的研究中，验证了由于EC中的SARS-COV-2峰值激活而导致的长期变化是必要的，例如，在涉及康复Covid-19患者的动物模型或临床研究中。 此外，未来的研究应考虑通过SARS-COV-2超出所指示的时间点的EC激活的后果，以及其他SARS-COV-2穗变异的血管炎症作用。 总之，我们的结果提供了SARS-COV-2的血管炎症作用的详细和全面表征。 我们表明内皮在确定Covid-19感染的结果时发挥着重要作用，例如血管炎症和系统性器官损伤，并且可能超出急性感染阶段。 治疗策略还应考虑SARS-COV-2对血管内皮的炎症作用的程度。 针对EC保护和预防内皮损伤的治疗对于Covid-19后遗症效果的预防和管理可能是必不可少的。 SARS-COV-2穗蛋白对人内皮细胞HTTPS://www.Researchsquare.com/Article/-5003230/v1的持续血管炎症作用，我没有令人愉快。 然而，我很感谢，在纠正中，那些从一开始阅读工作的人始终已知保护中内皮从尖峰蛋白质中普遍倾向。 衷心感谢您。 没有你的支持，我不能这样做。 我的研究仅由支付认购本食盒和直接捐赠资助。 目前（10/24/24），13.9K订阅者的存储器，只有364名是支付的用户。 如果您想捐赠加密，请给我发电子邮件。 如果您舒适地成为付费订阅者，请考虑这样做。 或者，请直接在https://wmcresearch.org/donate/捐赠

https://open.substack.com/pub/wmcresearch/p/gultom-et-al-proving-spike-protein